Speciation in Coastal Basins Driven by Staggered Headwater Captures: Dispersal of a Species Complex, Leporinus bahiensis, as Revealed by Genome-wide SNP Data.

Past sea level changes and geological instability along watershed boundaries have largely influenced fish distribution across coastal basins, either by dispersal via palaeodrainages now submerged or by headwater captures, respectively. Accordingly, the South American Atlantic coast encompasses several small and isolated drainages that share a similar species composition, representing a suitable model to infer historical processes. Leporinus bahiensis is a freshwater fish species widespread along adjacent coastal basins over narrow continental shelf with no evidence of palaeodrainage connections at low sea level periods. Therefore, this study aimed to reconstruct its evolutionary history to infer the role of headwater captures in the dispersal process. To accomplish this, we employed molecular-level phylogenetic and population structure analyses based on Sanger sequences (5 genes) and genome-wide SNP data. Phylogenetic trees based on Sanger data were inconclusive, but SNPs data did support the monophyletic status of L. bahiensis. Both COI and SNP data revealed structured populations according to each hydrographic basin. Species delimitation analyses revealed from 3 (COI) to 5 (multilocus approach) MOTUs, corresponding to the sampled basins. An intricate biogeographic scenario was inferred and supported by Approximate Bayesian Computation (ABC) analysis. Specifically, a staggered pattern was revealed and characterized by sequential headwater captures from basins adjacent to upland drainages into small coastal basins at different periods. These headwater captures resulted in dispersal throughout contiguous coastal basins, followed by deep genetic divergence among lineages. To decipher such recent divergences, as herein represented by L. bahiensis populations, we used genome-wide SNPs data. Indeed, the combined use of genome-wide SNPs data and ABC method allowed us to reconstruct the evolutionary history and speciation of L. bahiensis. This framework might be useful in disentangling the diversification process in other neotropical fishes subject to a reticulate geological history.

Reduced gene flow and bottleneck in the threatened giant armadillo (Priodontes maximus): implications for its conservation.

The progressive fragmentation and loss of habitats represent the main threats for endangered species, causing genetic consequences that may have potential implications for a population's long-term persistence. Large mammals are the most affected species among vertebrates. The giant armadillo Priodontes maximus is a large South American mammal threatened species, showing nocturnal, solitary and fossorial behavior, occurring at low population densities, and its population dynamics are still poorly known. In this study, we carried out the first assessment of genetic variability and population genetic structure of the species, using a panel of 15 polymorphic microsatellites developed by high-throughput genome sequencing. The spatial Bayesian clustering, Fst and Dest results indicated the presence of two genetic clusters (K = 2) in the study area. These results suggest a reduction in gene flow between individuals inhabiting the Brazilian savanna (Cerrado) and the Pantanal wetlands, with the increased human-driven habitat modifications possibly contributing for this scenario. A bottleneck signal was detected in both populations, and a subpopulation structuring in the Cerrado may also be reflecting consequences of the extensive habitat modifications. Findings from this study provide important and useful information for the future maintenance of genetic diversity and long-term conservation of this flagship species.

Genomic Signatures of Divergent Ecological Strategies in a Recent Radiation of Neotropical Wild Cats.

Ecological differentiation among diverging species is an important component of the evolutionary process and can be investigated in rapid and recent radiations. Here, we use whole genome sequences of five species from the genus Leopardus, a recently diversified Neotropical lineage with species bearing distinctive morphological, ecological, and behavioral features, to investigate genome-wide diversity, comparative demographic history and signatures of positive selection. Our results show that divergent ecological strategies are reflected in genomic features, for example a generalist species shows historically larger effective population size and higher heterozygosity than habitat specialists. The demographic history of these cats seems to have been jointly driven by climate fluctuations and habitat specialization, with different ecological adaptations leading to distinct trajectories. Finally, a gene involved in vertebrate retinal neurogenesis (POU4F2) was found to be under positive selection in the margay, a cat with notoriously large eyes that are likely associated with its nocturnal and arboreal specializations.

Genetic population structure of Pseudoplatystoma corruscans (Siluriformes: Pimelodidae) and evidence of temporal variation in structure.

Surubim (Pseudoplatystoma corruscans, Pimelodidae) are migratory catfish native to the rivers in the La Plata and São Francisco basins. They are piscivores that attain considerable body sizes and are a valuable economic resource. Surubim exhibits extensive migrations during its life cycle that may affect the population structure at vast geographic scales. The authors examined the genetic diversity and population genetic structure of P. corruscans using microsatellite markers from a comprehensive sampling of 260 individuals from the Upper and Lower Paraná River. They identified two well-differentiated genetic clusters corresponding to a natural geographic barrier historically separating Upper and Lower Paraná regions. They also demonstrated temporal variation in population genetic structure at a site in Lower Paraná close to the confluence with the Paraguay River, most likely explained by the influx of migrant fishes at certain times of the year.

Molecular phylogeny and species delimitation of the genus Schizodon (Characiformes, Anostomidae).

The genus Schizodon is part of a group of headstanders and relatives (Family Anostomidae) that are widespread and ecologically important fishes in South American rivers. Schizodon includes 15 nominal species but their taxonomy has been challenging due to paucity of decisive characters to diagnose species. We present new molecular data to assess species boundaries or molecular operational taxonomic units (MOTUs), and to infer phylogenetic relationships among species. Evidence from two mitochondrial and three nuclear genes was used in these analyses. Mitochondrial DNA data for 112 specimens (from 11 nominal species) supported 13 consensus MOTUs, six of which matched valid nominal species (Schizodon borellii, S. fasciatus, S. intermedius, S. isognathus, S. knerii and S. scotorhabdotus). The nominal species Schizodon vittatus, S. nasutus, and S. dissimilis were subdivided into two MOTUs each, revealing either cryptic species or strong population structuring. In contrast, S. platae and S. jacuiensis constituted a single MOTU, indicating a possible case of synonymy. Our phylogenetic analysis subdivided the genus Schizodon into two large clades that are compatible with observed color patterns and biogeographic distribution. The first clade includes species with three to four conspicuous dark vertical bars on the flanks that originated in the Amazonas region (S. borellii, S. dissimilis, S. intermedius, S. fasciatus, S. scotorhabdotus, S. vittatus, and a cryptic species, Schizodon aff. vittatus). The second clade includes species with a conspicuous dark caudal blotch on the caudal peduncle, with vertical bars absent or inconspicuous, with a biogeographic origin in the La Plata drainage (S. isognathus, S. jacuiensis, S. knerii, S. nasutus and S. platae). Our results reinforce the importance of using molecular analyses to accelerate the study of diversity, particularly in groups with a wide distribution, few variable meristic characters, and high morphological plasticity, which may hide still unknown or underestimated diversity.

A new genus of Anostomidae (Ostariophysi: Characiformes): Diversity, phylogeny and biogeography based on cytogenetic, molecular and morphological data.

A new genus of Anostomidae (Characiformes) is described to include ten valid extant species previously classified in Leporinus or Hypomasticus and distributed throughout most major river basins in South America: L. brinco, L. conirostris, L. elongatus, H. garmani, L. macrocephalus, L. muyscorum, L. obtusidens, L. piavussu, L. reinhardti, and L. trifasciatus. The monophyly of Megaleporinus is well-supported in a phylogenetic analysis based on two mitochondrial and three nuclear genes, as well as its sister group relationship to Abramites. Megaleporinus is diagnosed by having the exclusive combination of three unicuspid teeth on each premaxillary and dentary bone and a color pattern composed of one to four dark midlateral blotches. Additional distinguishing features and possible synapomorphies include a unique ZZ/ZW sex chromosome system confirmed for six congeners and a drumming apparatus wherein the first rib is elongated and associated with hypertrophied intercostal muscles, which was confirmed for three congeners as exclusive to mature males. Furthermore, our study identified at least four undescribed cryptic species, emphasizing the need for further taxonomic work and genetic analyses. A time-calibrated phylogenetic and biogeographical analysis of the new genus suggests that speciation in the proto-Amazon-Orinoco lineage was primarily driven by paleogeographic processes, such as the formation of the Orinoco and Tocantins basins. Dispersal and diversification of the genus in coastal basins draining the Eastern Brazilian Shield appears to have been facilitated by connections between paleo-basins during low sea level periods and headwater captures between coastal and inland watersheds. The present contribution demonstrates the importance of integrating data from morphology, DNA sequences and cytogenetics to advance the taxonomy and systematics of any complex species group.

Molecular analysis reveals hidden diversity in Zungaro (Siluriformes: Pimelodidade): a genus of giant South American catfish.

The genus Zungaro contains some of the largest catfish in South America. Two valid species are currently recognized: Zungaro jahu, inhabiting the Paraná and Paraguay basins, and Zungaro zungaro, occurring in the Amazonas and Orinoco basins. Analysing Zungaro specimens from the Amazonas, Orinoco, Paraguay and Paraná basins, based on the sequencing of COI and D-loop, we found at least three MOTUs, indicating the existence of hidden diversity within this fish group. Considering the ecological and economic values of this fish, our results are surely welcomed for its conservation, disclosing new findings on its diversity and pointing out the necessity for a detailed taxonomic revision.

DNA barcode and evolutionary relationship within Laemolyta Cope 1872 (Characiformes: Anostomidae) through molecular analyses.

The Laemolyta genus is a monophyletic group with five valid species. Phylogenetic relationships among the species of this genus are unknown. We analyzed four nominal Laemolyta species. The COI gene for all individuals was amplified and the genetic distances were estimated. We performed genetic distance analyses to determine the different MOTUs. Two mitochondrial (COI and CytB) and three nuclear (Myh6, RAG1 and RAG2) markers were amplified for one individual of each identified MOTU. Maximum Parsimony and Maximum Likelihood were conducted using concatenate alignment. In addition, multilocus Bayesian species tree was carried out. By using DNA barcode, we identified six different MOTUs. The COI inter-MOTU distances ranged from 0.92% to 5.76%. The normalized mean intra-MOTU distance was 0.13%. The DNA barcode was useful to diagnose all species. Two clades showing distinct color patterns were recovered in all molecular phylogenetic trees. Clade A joined fishes with no vertical bars (L. garmani, L. taeniata 1 and L. taeniata 2) and clade B, fishes with vertical dark bars (L. fernandezi Araguaia, L. fernandezi Xingu, and L. proxima). The results were able to identify the cryptic biodiversity within the group and obtained the most complete Laemolyta phylogeny.

Wide-range genetic connectivity of Coney, Cephalopholis fulva (Epinephelidae), through oceanic islands and continental Brazilian coast.

The Epinephelidae form a group of species of high biological and economical interests. It´s phylogeographic patterns are not well known especially the distributed populations in the western region of the Atlantic Ocean. Among the representatives is a small species called Cephalopholis fulva, Coney, which presents a wide geographical distribution, polychromia, hermaphroditism and is quickly becoming a large target for the exploration of commercial fishing. The genetic and historical demography were obtained through the partial sequence analysis of Control Region from six locations on the coastline of Brazil from the northeast coast to the southwest coast, including the oceanic islands of Rocas Atoll and Fernando de Noronha Archipelago. The analyzed samples revealed a high genetic variability and a strong gene flow among the sampled locations. Additionally, the genetic data revealed that population expansions probably occurred due to the changes in the sea levels that occurred during the Pleistocene. The large population connectivity found in Coney constitutes relevant conditions for their biological conservation.

Testing and optimizing metabarcoding of iDNA from dung beetles to sample mammals in the hyperdiverse Neotropics.

Over the past few years, insects have been used as samplers of vertebrate diversity by assessing the ingested-derived DNA (iDNA), and dung beetles have been shown to be a good mammal sampler given their broad feeding preference, wide distribution and easy sampling. Here, we tested and optimized the use of iDNA from dung beetles to assess the mammal community by evaluating if some biological and methodological aspects affect the use of dung beetles as mammal species samplers. We collected 403 dung beetles from 60 pitfall traps. iDNA from each dung beetle was sequenced by metabarcoding using two mini-barcodes (12SrRNA and 16SrRNA). We assessed whether dung beetles with different traits related to feeding, nesting and body size differed in the number of mammal species found in their iDNA. We also tested differences among four killing solutions in preserving the iDNA and compared the effectiveness of each mini barcode to recover mammals. We identified a total of 50 mammal OTUs (operational taxonomic unit), including terrestrial and arboreal species from 10 different orders. We found that at least one mammal-matching sequence was obtained from 70% of the dung beetle specimens. The number of mammal OTUs obtained did not vary with dung beetle traits as well as between the killing solutions. The 16SrRNA mini-barcode recovered a higher number of mammal OTUs than 12SrRNA, although both sets were partly non-overlapping. Thus, the complete mammal diversity may not be achieved by using only one of them. This study refines the methodology for routine assessment of tropical mammal communities via dung beetle 'samplers' and its universal applicability independently of the species traits of local beetle communities.

Population genomics reveals how 5 ka of human occupancy led the Lima leaf-toed gecko (Phyllodactylus sentosus) to the brink of extinction.

Small species with high home fidelity, high ecological specialization or low vagility are particularly prone to suffer from habitat modification and fragmentation. The Lima leaf-toed gecko (Phyllodactylus sentosus) is a critically endangered Peruvian species that shelters mostly in pre-Incan archeological areas called huacas, where the original environmental conditions are maintained. We used genotyping by sequencing to understand the population genomic history of P. sentosus. We found low genetic diversity (He 0.0406-0.134 and nucleotide diversity 0.0812-0.145) and deviations of the observed heterozygosity relative to the expected heterozygosity in some populations (Fis - 0.0202 to 0.0187). In all analyses, a clear population structuring was observed that cannot be explained by isolation by distance alone. Also, low levels of historical gene flow were observed between most populations, which decreased as shown in contemporary migration rate analysis. Demographic inference suggests these populations experienced bottleneck events during the last 5 ka. These results indicate that habitat modification since pre-Incan civilizations severely affected these populations, which currently face even more drastic urbanization threats. Finally, our predictions show that this species could become extinct in a decade without further intervention, which calls for urgent conservation actions being undertaken.

Comparing iDNA from mosquitoes and flies to survey mammals in a semi-controlled Neotropical area.

Ingested-derived DNA (iDNA) from insects represents a powerful tool for assessing vertebrate diversity because insects are easy to sample, have a diverse diet and are widely distributed. Because of these advantages, the use of iDNA for detecting mammals has gained increasing attention. Here we aimed to compare the effectiveness of mosquitoes and flies to detect mammals with a small sampling effort in a semi-controlled area, a zoo that houses native and non-native species. We compared mosquitoes and flies regarding the number of mammal species detected, the amount of mammal sequence reads recovered, and the flight distance range for detecting mammals. We also verified if the combination of two mini-barcodes (12SrRNA and 16SrRNA) would perform better than either mini-barcode alone to inform local mammal biodiversity from iDNA. To capture mosquitoes and flies, we distributed insect traps in eight sampling points during 5 days. We identified 43 Operational Taxonomic Units from 10 orders, from the iDNA of 17 mosquitoes and 46 flies. There was no difference in the number of species recovered per individual insect between mosquitoes and flies, but the number of flies captured was higher, resulting in more mammal species recovered by flies. Eight species were recorded exclusively by mosquitoes and 20 by flies, suggesting that using both samplers would allow a more comprehensive screening of the biodiversity. The maximum distance recorded was 337 m for flies and 289 m for mosquitoes, but the average range distance did not differ between insect groups. Our assay proved to be efficient for mammal detection, considering the high number of species detected with a reduced sampling effort.

Efficiency of eDNA and iDNA in assessing vertebrate diversity and its abundance.

Environmental DNA (eDNA) and invertebrate-derived DNA (iDNA) have been increasingly recognized as powerful tools for biodiversity assessment and conservation management. However, eDNA/iDNA efficiency for vertebrate diversity assessment remains uncertain, and comparisons to conventional methods are still rare. Through a meta-analysis of previously published vertebrate diversity surveys, we compared the efficiency of eDNA/iDNA against conventional methods across several types of samplers, vertebrate groups, and locations (tropical vs. temperate zones). We also assess eDNA/iDNA efficiency to estimate relative abundance or biomass over different molecular methods (qPCR and metabarcoding) and type of experiment (in the laboratory or in the field). We showed that for water sampler, fish as a target species, and studies achieved in temperate zones, eDNA presents lower risk of not detecting a species or a site with a target species than conventional methods. These results show that eDNA is an efficient tool to assess fish diversity. Moreover, eDNA data presents positive correlation with fish abundance or biomass. However, such correlation was higher in laboratory experiments than in the field. For the other samplers, vertebrate groups, and in tropical zones we were not able to draw general conclusion, highlighting the urgency of conducting more comparative studies.

Hidden diversity in Prochilodus nigricans: A new genetic lineage within the Tapajós River basin.

Highly spread through the Amazon River basin, Prochilodus nigricans have had its taxonomic validity recently questioned, when genetic differences between Western and Eastern Amazon populations from the Brazilian shield were detected. This area has been seeing as a region of high ichthyofaunal diversity and endemism, in which the hybrid origin of the Tapajós River basin has been raised. In this paper, we report a new molecular lineage within P. nigricans of Tapajós River, highlighting this region still hides taxonomically significant diversity. Haplotype networks were reconstructed using the mitochondrial COI and ATP6/8 markers, which were also used to calculate genetic distances among clusters. We additionally conducted a delimiting species approach by employing a Generalized Mixed Yule-Coalescent model (GMYC) with COI sequences produced here, and previous ones published for individuals sampled across the Amazon River basin. In addition to the genetic differentiation within P. nigricans, our findings favor the hypothesis of hybrid origin of the Tapajós River basin and reaffirm the importance of studies aiming to investigate hidden diversity to address taxonomic and biogeographic issues, that certainly benefit better biodiversity conservation actions.

DNA mini-barcoding of leporids using noninvasive fecal DNA samples and its significance for monitoring an invasive species.

Introduced in South America at the end of the 19th century, the European hare population has expanded dramatically and now represents a risk to native Brazilian forest rabbits. Monitoring the invasive Lepus europaeus and its coexistence with native Sylvilagus brasiliensis is a challenge that can be efficiently addressed by the use of molecular tools. This work describes a set of primers useful for amplifying three mini-barcodes for the molecular identification of both invasive and native leporid species using degraded fecal DNA. In addition, tests in silico indicate that these mini-barcodes can successfully amplify the DNA sequences of a number of leporids. These mini-barcodes constitute a powerful tool for the monitoring and management of the invasive L. europaeus and the conservation of native rabbits.

Molecular Evidences of a Hidden Complex Scenario in Leporinus cf. friderici.

The megadiversity of the neotropical ichthyofauna has been associated to recent diversification processes, reflecting in subtle or lacking morphological differentiation between species, challenging the classical taxonomic identification. Leporinus friderici occurs in several river basins of South America, and its nominal taxonomic validity has been questioned. Its wide distribution within the Brazilian Shield suggests that this species could be genetically structured among the hydrographic basins, despite a sharp morphological similarity. In this study, we performed phylogenetic analyses, based on three nuclear (recombination activating gene 1, RAG1, recombination activating gene 2, RAG2, and myosin heavy chain 6 cardiac muscle alpha gene, Myh6) and two mitochondrial (COI and Cytochrome b, Cytb) markers, in specimens morphologically similar to L. friderici and related species from different hydrographic basins in South America. Our phylogenetic tree identified four well-supported clades, which point out to the existence of taxonomic inconsistencies within this fish group. A clade named L. cf. friderici sensu stricto included eight Molecular Operational Taxonomic Units recently diversified in the Brazilian Shield basins. These results were also confirmed by a single-gene species delimitation analysis. It is suggested that this clade includes a species complex, characterizing taxonomic uncertainties. Another clade recovered only L. friderici from the Suriname rivers, validating this nominal species in its type locality. A third no-named clade, characterized by deeper species divergence, recovered five different nominal species interleaved with other undescribed forms previously also recognized as L. cf. friderici, indicating taxonomic errors. The fourth clade only included L. taeniatus. Our results showed a complex scenario involving the morphotype L. cf. friderici and allowed us to address aspects related to evolutionary diversification of this fish group and historical processes involved with, highlighting the importance of revealing hidden biodiversity for the taxonomy and conservationist action plans of these fish.

Genetic Pattern and Demographic History of Salminus brasiliensis: Population Expansion in the Pantanal Region during the Pleistocene.

Pleistocene climate changes were major historical events that impacted South American biodiversity. Although the effects of such changes are well-documented for several biomes, it is poorly known how these climate shifts affected the biodiversity of the Pantanal floodplain. Fish are one of the most diverse groups in the Pantanal floodplains and can be taken as a suitable biological model for reconstructing paleoenvironmental scenarios. To identify the effects of Pleistocene climate changes on Pantanal's ichthyofauna, we used genetic data from multiple populations of a top-predator long-distance migratory fish, Salminus brasiliensis. We specifically investigated whether Pleistocene climate changes affected the demography of this species. If this was the case, we expected to find changes in population size over time. Thus, we assessed the genetic diversity of S. brasiliensis to trace the demographic history of nine populations from the Upper Paraguay basin, which includes the Pantanal floodplain, that form a single genetic group, employing approximate Bayesian computation (ABC) to test five scenarios: constant population, old expansion, old decline, old bottleneck following by recent expansion, and old expansion following by recent decline. Based on two mitochondrial DNA markers, our inferences from ABC analysis, the results of Bayesian skyline plot, the implications of star-like networks, and the patterns of genetic diversity (high haplotype diversity and low-to-moderate nucleotide diversity) indicated a sudden population expansion. ABC allowed us to make strong quantitative inferences about the demographic history of S. brasiliensis. We estimated a small ancestral population size that underwent a drastic fivefold expansion, probably associated with the colonization of newly formed habitats. The estimated time of this expansion was consistent with a humid and warm phase as inferred by speleothem growth phases and travertine records during Pleistocene interglacial periods. The strong concordance between our genetic inferences and this historical data could represent the first genetic record of a humid and warm phase in the Pantanal in the period since the Last Interglacial to 40 ka.

Comparing hair-morphology and molecular methods to identify fecal samples from Neotropical felids.

To avoid certain problems encountered with more-traditional and invasive methods in behavioral-ecology studies of mammalian predators, such as felids, molecular approaches have been employed to identify feces found in the field. However, this method requires a complete molecular biology laboratory, and usually also requires very fresh fecal samples to avoid DNA degradation. Both conditions are normally absent in the field. To address these difficulties, identification based on morphological characters (length, color, banding, scales and medullar patterns) of hairs found in feces could be employed as an alternative. In this study we constructed a morphological identification key for guard hairs of eight Neotropical felids (jaguar, oncilla, Geoffroy's cat, margay, ocelot, Pampas cat, puma and jaguarundi) and compared its efficiency to that of a molecular identification method, using the ATP6 region as a marker. For this molecular approach, we simulated some field conditions by postponing sample-conservation procedures. A blind test of the identification key obtained a nearly 70% overall success rate, which we considered equivalent to or better than the results of some molecular methods (probably due to DNA degradation) found in other studies. The jaguar, puma and jaguarundi could be unequivocally discriminated from any other Neotropical felid. On a scale ranging from inadequate to excellent, the key proved poor only for the margay, with only 30% of its hairs successfully identified using this key; and have intermediate success rates for the remaining species, the oncilla, Geoffroy's cat, ocelot and Pampas cat, were intermediate. Complementary information about the known distributions of felid populations may be necessary to substantially improve the results obtained with the key. Our own molecular results were even better, since all blind-tested samples were correctly identified. Part of these identifications were made from samples kept in suboptimal conditions, with some samples remaining outdoors for up to seven days, simulating conditions in the field. It appears that both methods can be used, depending on the available laboratory facilities and on the expected results.

Revealing Hidden Diversity of the Underestimated Neotropical Ichthyofauna: DNA Barcoding in the Recently Described Genus Megaleporinus (Characiformes: Anostomidae).

Molecular studies have improved our knowledge on the neotropical ichthyofauna. DNA barcoding has successfully been used in fish species identification and in detecting cryptic diversity. Megaleporinus (Anostomidae) is a recently described freshwater fish genus within which taxonomic uncertainties remain. Here we assessed all nominal species of this genus using a DNA barcode approach (Cytochrome Oxidase subunit I) with a broad sampling to generate a reference library, characterize new molecular lineages, and test the hypothesis that some of the nominal species represent species complexes. The analyses identified 16 (ABGD and BIN) to 18 (ABGD, GMYC, and PTP) different molecular operational taxonomic units (MOTUs) within the 10 studied nominal species, indicating cryptic biodiversity and potential candidate species. Only Megaleporinus brinco, Megaleporinus garmani, and Megaleporinus elongatus showed correspondence between nominal species and MOTUs. Within six nominal species, a subdivision in two MOTUs was found, while Megaleporinus obtusidens was divided in three MOTUs, suggesting that DNA barcode is a very useful approach to identify the molecular lineages of Megaleporinus, even in the case of recent divergence (< 0.5 Ma). Our results thus provided molecular findings that can be used along with morphological traits to better define each species, including candidate new species. This is the most complete analysis of DNA barcode in this recently described genus, and considering its economic value, a precise species identification is quite desirable and fundamental for conservation of the whole biodiversity of this fish.

A biodiversity hotspot losing its top predator: The challenge of jaguar conservation in the Atlantic Forest of South America.

The jaguar is the top predator of the Atlantic Forest (AF), which is a highly threatened biodiversity hotspot that occurs in Brazil, Paraguay and Argentina. By combining data sets from 14 research groups across the region, we determine the population status of the jaguar and propose a spatial prioritization for conservation actions. About 85% of the jaguar's habitat in the AF has been lost and only 7% remains in good condition. Jaguars persist in around 2.8% of the region, and live in very low densities in most of the areas. The population of jaguars in the AF is probably lower than 300 individuals scattered in small sub-populations. We identified seven Jaguar Conservation Units (JCUs) and seven potential JCUs, and only three of these areas may have ≥50 individuals. A connectivity analysis shows that most of the JCUs are isolated. Habitat loss and fragmentation were the major causes for jaguar decline, but human induced mortality is the main threat for the remaining population. We classified areas according to their contribution to jaguar conservation and we recommend management actions for each of them. The methodology in this study could be used for conservation planning of other carnivore species.