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      Fifty years after: A taxonomic revision of the amphibian species from the Ecuadorian biodiversity hotspot Abra de Zamora, with description of two new Pristimantis species

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          Abstract

          Abra de Zamora is an important biodiversity hotspot in southern Ecuador. Between 1938 and 2010, eleven species of frogs were described from here: Lynchius flavomaculatus, Gastrotheca psychrophila, Pristimantis balionotus, P. colodactylus, P. cryptomelas, P. percultus, P. versicolor, P. vidua, Telmatobius cirrhacelis, P. andinognomus, and Atelopus podocarpus. Unfortunately, many of these species were not re-encountered after their original description, and for the majority DNA samples were not available, making their phylogenetic position unknown. In this study, we assess the current state of the amphibians from Abra de Zamora, by: i. redescribing the species which were first reported from the area, by contributing genetic delimitation (for L. flavomaculatus, P. balionotus, P. colodactylus, P. percultus, and P. vidua), release call ( L. flavomaculatus) and advertisement call descriptions (for P. balionotus, P. vidua and P. versicolor); ii. presenting an updated amphibian species list of Abra de Zamora, with the description of two additional Pristimantis species; iii. updating the distribution of these species, including data collected in similar montane habitats from surrounding areas; and iv. amending recommendations regarding their conservation status.

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          The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice.

          Vocalizations of anuran amphibians have received much attention in studies of behavioral ecology and physiology, but also provide informative characters for identifying and delimiting species. We here review the terminology and variation of frog calls from a perspective of integrative taxonomy, and provide hands-on protocols for recording, analyzing, comparing, interpreting and describing these sounds. Our focus is on advertisement calls, which serve as premating isolation mechanisms and, therefore, convey important taxonomic information. We provide recommendations for terminology of frog vocalizations, with call, note and pulse being the fundamental subunits to be used in descriptions and comparisons. However, due to the complexity and diversity of these signals, an unequivocal application of the terms call and note can be challenging. We therefore provide two coherent concepts that either follow a note-centered approach (defining uninterrupted units of sound as notes, and their entirety as call) or a call-centered approach (defining uninterrupted units as call whenever they are separated by long silent intervals) in terminology. Based on surveys of literature, we show that numerous call traits can be highly variable within and between individuals of one species. Despite idiosyncrasies of species and higher taxa, the duration of calls or notes, pulse rate within notes, and number of pulses per note appear to be more static within individuals and somewhat less affected by temperature. Therefore, these variables might often be preferable as taxonomic characters over call rate or note rate, which are heavily influenced by various factors. Dominant frequency is also comparatively static and only weakly affected by temperature, but depends strongly on body size. As with other taxonomic characters, strong call divergence is typically indicative of species-level differences, whereas call similarities of two populations are no evidence for them being conspecific. Taxonomic conclusions can especially be drawn when the general advertisement call structure of two candidate species is radically different and qualitative call differences are thus observed. On the other hand, quantitative differences in call traits might substantially vary within and among conspecific populations, and require careful evaluation and analysis. We provide guidelines for the taxonomic interpretation of advertisement call differences in sympatric and allopatric situations, and emphasize the need for an integrative use of multiple datasets (bio-acoustics, morphology, genetics), particularly for allopatric scenarios. We show that small-sized frogs often emit calls with frequency components in the ultrasound spectrum, although it is unlikely that these high frequencies are of biological relevance for the majority of them, and we illustrate that detection of upper harmonics depends also on recording distance because higher frequencies are attenuated more strongly. Bioacoustics remains a prime approach in integrative taxonomy of anurans if uncertainty due to possible intraspecific variation and technical artifacts is adequately considered and acknowledged.
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            Underestimation of Species Richness in Neotropical Frogs Revealed by mtDNA Analyses

            Background Amphibians are rapidly vanishing. At the same time, it is most likely that the number of amphibian species is highly underestimated. Recent DNA barcoding work has attempted to define a threshold between intra- and inter-specific genetic distances to help identify candidate species. In groups with high extinction rates and poorly known species boundaries, like amphibians, such tools may provide a way to rapidly evaluate species richness. Methodology Here we analyse published and new 16S rDNA sequences from 60 frog species of Amazonia-Guianas to obtain a minimum estimate of the number of undescribed species in this region. We combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs. Principal Findings In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contrary to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species. Conclusions Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas. Significance As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised.
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              Global Conservation Significance of Ecuador's Yasuní National Park

              Background The threats facing Ecuador's Yasuní National Park are emblematic of those confronting the greater western Amazon, one of the world's last high-biodiversity wilderness areas. Notably, the country's second largest untapped oil reserves—called “ITT”—lie beneath an intact, remote section of the park. The conservation significance of Yasuní may weigh heavily in upcoming state-level and international decisions, including whether to develop the oil or invest in alternatives. Methodology/Principal Findings We conducted the first comprehensive synthesis of biodiversity data for Yasuní. Mapping amphibian, bird, mammal, and plant distributions, we found eastern Ecuador and northern Peru to be the only regions in South America where species richness centers for all four taxonomic groups overlap. This quadruple richness center has only one viable strict protected area (IUCN levels I–IV): Yasuní. The park covers just 14% of the quadruple richness center's area, whereas active or proposed oil concessions cover 79%. Using field inventory data, we compared Yasuní's local (alpha) and landscape (gamma) diversity to other sites, in the western Amazon and globally. These analyses further suggest that Yasuní is among the most biodiverse places on Earth, with apparent world richness records for amphibians, reptiles, bats, and trees. Yasuní also protects a considerable number of threatened species and regional endemics. Conclusions/Significance Yasuní has outstanding global conservation significance due to its extraordinary biodiversity and potential to sustain this biodiversity in the long term because of its 1) large size and wilderness character, 2) intact large-vertebrate assemblage, 3) IUCN level-II protection status in a region lacking other strict protected areas, and 4) likelihood of maintaining wet, rainforest conditions while anticipated climate change-induced drought intensifies in the eastern Amazon. However, further oil development in Yasuní jeopardizes its conservation values. These findings form the scientific basis for policy recommendations, including stopping any new oil activities and road construction in Yasuní and creating areas off-limits to large-scale development in adjacent northern Peru.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: VisualizationRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 September 2020
                2020
                : 15
                : 9
                : e0238306
                Affiliations
                [1 ] Museo de Zoología, Universidad Técnica Particular de Loja, Loja, Ecuador
                [2 ] Laboratorio de Ecología Tropical y Servicios Ecosistémicos - EcoSs Lab, Departamento de Ciencias Biológicas, Universidad Técnica Particular de Loja, Loja, Ecuador
                [3 ] Biotechnology Center (BIOTEC), Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden (TUD), Dresden, Germany
                [4 ] Programa de Doctorado en Conservación de Recursos Naturales, Escuela Internacional de Doctorado, Universidad Rey Juan Carlos, Móstoles, Madrid, Spain
                [5 ] Faculty of Natural and Agricultural Sciences, Ovidius University Constanța, Constanța, Romania
                Universitat Trier, GERMANY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-9252-1596
                http://orcid.org/0000-0002-8866-4316
                http://orcid.org/0000-0002-4593-1728
                Article
                PONE-D-20-15050
                10.1371/journal.pone.0238306
                7482940
                36e6edc2-8fab-4654-a062-5b611c495d99
                © 2020 Székely et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 May 2020
                : 12 August 2020
                Page count
                Figures: 29, Tables: 3, Pages: 59
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100013724, Critical Ecosystem Partnership Fund;
                Award ID: CEPF-108984
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004630, Mohammed bin Zayed Species Conservation Fund;
                Award ID: 172515484
                Award Recipient :
                This study was funded by the Critical Ecosystem Partnership Fund (CEPF), grant CEPF-108984, through the “Amphibian Conservation in the Abra de Zamora Key Biodiversity Area of Ecuador” project. Fieldwork and research of marsupial frogs was supported by the Mohamed bin Zayed Species Conservation Fund, through project 172515484 "Conservation of the Ridge Marsupial Frog (Gastrotheca psychrophila) in southern Ecuador". DS benefited from a research grant from Universidad Técnica Particular de Loja (Convocatoria Estancias Cortas de Investigación 2019).
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                DNA matrices are available in Zenodo: https://doi.org/10.5281/zenodo.3952997.

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