**5. Conclusions**

A 5 × 200-m progressively increasing speed test and the determination of V4 calculated by interpolation of a second-order polynomial function fitted to the swimming speed vs. lactate concentration data seem to provide physiological and biomechanical variables with a large interindividual variability. It should be expected that swimmers will adjust their mechanics at increased metabolic cost to maintain the required speed during an intermittent training set including 400-m repetitions. Specifically, increased SR and decreased SL may be observed when swimmers aim to maintain speed during a 5 × 400-m intermittent training set. It seems that swimmers change the arm stroke profile in aiming to maintain the required speed during a long-duration constant intensity set. The large interindividual variation between swimmers, possibly because of their specialty or the characteristics (i.e., number of repetitions, interval time, distance) of the test used to calculate the required parameters, should be considered. Coaches should be aware that prescribed physiological or mechanical parameters may be altered when swimmers follow a training pace corresponding to V4 during aerobic endurance training.

**Author Contributions:** Conceptualization, A.G.T. and G.G.A.; methodology, A.G.T., P.G.B., G.G.A.; software, G.G.A.; validation, A.G.T. and P.G.B.; formal analysis, P.G.B.; investigation, G.G.A., I.S.N. and I.M.; resources, A.G.T.; data curation G.G.A.; writing – original draft preparation, G.G.A.; writing—review and editing A.G.T.; visualization, P.G.B.; supervision, A.G.T.; project administration, A.G.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank the swimmers for their participation in the study.

**Conflicts of Interest:** The authors declare no conflict of interest, financial or otherwise.


**6. Conservation**

Evaluating the conservation status and conservation needs of amphibian species is extremely challenging. Traditional threats (e.g., habitat destruction, water contamination, pesticides) and the emergence of novel factors (e.g., climate change, introduced species, and emerging diseases) need to be combined when developing conservation strategies [23,76,124,270]. With all these considerations in mind, we present our evaluation of the conservation of each Ecuadorian glassfrog in Table S7. The most endangered species are: *Centrolene buckleyi*, *C*. *charapita*, *C*. *geckoidea*, *C*. *medemi*, *C*. *pipilata*, *Cochranella mache*, *Nymphargus balionotus*, *N*. *manduriacu*, *N*. *megacheirus*, and *N*. *sucre* (Table S7) and, therefore, are most likely to go extinct because of any of the aforementioned variables. Additionally, numerous species are *Data Deficient* (Table S7) and urgently require additional research.

The most conspicuous and immediate threat to glassfrog conservation is habitat destruction, which is rampant in the Chocó ecoregion (Esmeraldas Province) and also on the northwestern slopes of the Andes (Figure 18). The Chocó suffers the highest deforestation rate in Ecuador, thus, affecting all the species that occur there. Five Chocoan glassfrogs (*H*. *fleischmanni*, *C*. *geckoidea*, *T*. *pulverata*, *T*. *spinosa*, *Cochranella mache*) have more than half of their potential distribution affected by human activities (Table S4).

Habitat preservation is the most effective mechanism to protect diversity. However, we first need to identify the areas that should be prioritized for such actions. As a first approach, it is clear that both at the South American and Ecuadorian scales, mountain ranges harbor the highest density of species and, also, species that tend to have small distributions. Also, as shown in Figure 230, the Northern Andes are, by far, the most important biogeographic regions in terms of glassfrog species richness. Then, any effort directed towards the conservation of Andean forests will greatly benefit glassfrogs.

In Ecuador, more specifically, when the potential distributions of all glassfrog species are combined, we are able to identify areas with both high diversity and low human disturbance that are not included in the current system of protected areas of Ecuador (Figure 237). Based on this information, specifically, we recommend: (i) The implementation of a biological corridor between the National Parks of Cayambe-Coca and Sumaco, (ii) creation of a new protected area in the lower montane evergreen forest of the western Andes in Pichincha Province, and (iii) protection of the endemic Chocoan forest (threatened mainly by wood extraction and palm plantations). The aforementioned conservation actions agree with the recommendations produced by broader studies [76,388], in terms of the areas that need to be protected in Ecuador given the current threats and biodiversity patterns (Figures 238 and 239).

Other conservation actions should be directed towards species that are known from few localities that lie within areas that are (or will be) affected by human activities; this is the case, for example, for *Centrolene condor*, species endemic to the Cordillera del Cóndor, where hundreds of hectares have been conceded for mining activities [389]. A similar situation applies to *N*. *manduriacu*, known from a single locality (Reserva Río Manduriacu) that is concessioned for mining [21].

One of the novel threats to amphibian diversity worldwide is the disease caused by the fungus *Batrachochytrium dendrobatidis* (*Bd*), known as chytridiomycosis [23,390,391]. Chytridiomycosis has been implicated in the extinction of numerous Andean species, mainly harlequin toads (*Atelopus*) [31,33,392]. In Ecuador, ecological modeling predicts that the highest suitability for chytridiomycosis is in the Andes, at elevations above 2000 m [393]. The impact of the disease on Ecuadorian glassfrogs has not been assessed, but preliminary studies show that *Bd* has a relatively high prevalence in several species of Andean glassfrogs [92]. Because of the absence of long-term monitoring of amphibian populations in Ecuador, there is no certainty of the effect of *Bd* on most amphibians, but in places where *Bd* is present, it is possible that all vulnerable species are already extinct. Glassfrogs that persist in spite of infections are probably resistant to the disease or are exposed to a less-pathogenic strains of the fungus [93]. The disappearance of species such as *Nymphargus balionotus* and *Centrolene geckoidea* from relatively pristine areas may be related to chytridiomycosis.

**Figure 237.** Glassfrog species diversity in Ecuador. (**A**) Species diversity simplified into three categories: High, medium, and low. (**B**) Map showing protected areas in Ecuador and, in dark, regions that combine high glassfrog diversity and low impact by human activities. (**C**) Map showing protected areas in Ecuador and, in dark, regions with both medium diversity and low impact by human activities.

Another specific conservation threat to Andean species that depend on rivers and streams is the introduced rainbow trout, *Oncorhynchus mykiss* [124,394]. Laboratory experiments on the interaction of this exotic fish and tadpoles of the red-spotted glassfrog (*Nymphargus grandisonae*) show that trout prey on tadpoles and that its presence results in an increased mortality and phenotypic change [124]. Most likely, similar effects are produced on other amphibian species with aquatic larvae. Given that the rainbow trout has been introduced into most Ecuadorian Andean rivers and lakes, the only option to reduce its impact is to start eradication programs, at least in protected areas and wherever the trout might be threatening endangered amphibians [124]. Other studies suggest that climate change, chytridiomycosis, and their synergetic effects, likely represent the major threats that amphibians face in Ecuador [393] and worldwide [395].

Finally, climate change represents one of the most challenging conservations phenomenon for biodiversity, given its global scale and potentially large effect even on species that are found in relatively pristine areas. It has been shown that the same variables that explain the high levels of tropical diversity (e.g., narrow thermal tolerance and low dispersal) also make tropical species more vulnerable to rapid thermal change [396]. The thermal breath of tropical amphibians is poorly known, but it can be correlated with their elevational distribution; glassfrogs show, in general, a narrow elevational range (mean = 653 m ± 526; *n* = 150 species; Table S1), meaning that their optimal thermal niches are only available at very restricted elevations. The survival of a species depends, then, on its ability to disperse at a pace fast enough to find itself in a suitable climatic environment. Dispersion will also depend on factors that are extrinsic to the organism, such as habitat continuity. In areas where fragmentation is severe, species will not be able to shift their distributional ranges. From a conservation perspective, it is critical to have areas with elevational gradients and both terrestrial and riparian corridors to allow species' movements. Alternatively, in fragmented landscapes, assisted dispersal might represent a necessary conservation action [396].

**Figure 238.** Human impact on ecosystems of Ecuador.

**Figure 239.** Environmental risk and conservation priorities in Ecuador. (**A**) Environmental risk surface for continental Ecuador. This surface takes into account information on roads, human population density, airports, dams, agriculture and husbandry, and oil and mining industry [73]. (**B**) Conservation priorities in Ecuador; this map was constructed using the potential distribution of 809 species (amphibians, birds, mammals, plants), combined with feasibility of conservation [73].

The alternative solution for species is to adapt to the novel climatic conditions associated with anthropogenic climate change; however, rates of climatic niche change among populations of plants and animals are dramatically slower than projected rates of future climate change. This means that, most likely, populations may not be able to change their climatic niches rapidly enough to keep pace with changing conditions as global climate warms, with dispersal being the only venue to avoid extinction [397,398].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/6/222/s1, Table S1: Taxonomy, biogeographic distribution, and elevation of all recognized species of glassfrogs, Table S2: Taxon and genetic markers used in this study. Sequences generated in previous studies were downloaded from GenBank. Newly generated sequences are in bold blue, Table S3: Summary statistics for each marker used in the phylogenetic analyses, Table S4: Potential distribution of glassfrog species in Ecuador, with percentage affected by human activities, Table S5: Area Under Curve (AUC) values for potential distribution models, Table S6: Candidate species recognized in this study, Table S7: Conservation status of Ecuadorian glassfrogs.

**Author Contributions:** J.M.G. and D.F.C.-H. conceived the study; J.M.G. wrote the manuscript, with contributions from all authors; J.M.G., D.F.C.-H., P.P., R.W.M., and C.R.H. collected the data; J.M.G., C.R.H., and P.P. analyzed the data; J.M.G. and C.R.H. rendered all figures and tables. All authors have read and agreed to the published version of the manuscript.

**Funding:** JMG's research was supported by the National Science Foundation (DEB-1046408, DEB-1045960, DEB-1045991DEB–0608011, EF–0334928, DEB-1046408); Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación de Ecuador (PIC-08-0000470); Partnerships for Enhanced Engagement in Research Science [grant number P1-108]; JRS Biodiversity Foundation; the American Philosophical Society through the Lewis and Clark Fund for Exploration and Field Research; IUCN-Save Our Species; Pontificia Universidad Católica del Ecuador; Mashpi Biodiversity Reserve, Panorama Society Grant and Harris Scholarship Award of the University of Kansas Natural History Museum; Universidad Tecnológica Indoamérica; and Universidad San Francisco de Quito (Collaboration Grants 11164, 16871; COCIBA Grants 5521, 5467, 16808). DFCH's research was supported by María Elena Heredia, Laura Heredia, the Smithsonian Women's Committee, the 2002 Research Training Program, National Museum of Natural History, Smithsonian Institution, Tiputini Biodiversity Station, Instituto de Ecología Aplicada ECOLAP-USFQ, Río Guajalito Protected Forest, Mashpi Protected Forest/Metropolitan Tourists, Fundación Futuro, the Global Amphibian Assessment, Conservation International, the Atelopus Initiative, the Research Analysis Network for Neotropical Amphibians (RANA, supported by the National Science Foundation DEB-0130273), the Russel E. Train Education for Nature Program, World Wildlife Fund WWF, Programa "Becas de Excelencia", Secretaría de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT), Ecuador, and Universidad San Francisco de Quito USFQ (Chancellor grants, COCIBA grants, Collaboration grants, projects HUBI ID 34, 36, 39, 48, 1057, 7703, 12253, 13524, 16953). Work by JMG and DFCH was supported by "Proyecto Descubre Napo", an initiative of Universidad San Francisco de Quito in association with Wildlife Conservation Society and funded by the Gordon and Betty Moore Foundation as part of the project: WCS Consolidating Conservation of Critical Landscapes (mosaics) in the Andes.

**Acknowledgments:** This study took a very long time to write and review; it was supposed to be part of JMG's PhD dissertation but, instead, it took 12 additional years of punctuated work and the creative input of all coauthors, mainly CRH. There are numerous people to thank and, for sure, we will forget to mention some (we apologize in advance). This article was greatly improved by discussions with Santiago Castroviejo-Fisher, Marco Rada, Jesse Delia, Alessandro Catenazzi, Rudolf von May, Annabelle Wang, Elisa Bonaccorso, and three anonymous reviewers. We are grateful to the many individuals and institutions who provided specimens, tissues, calls, photos, and information included in this study: Luis A. Coloma, Ítalo Tapia (Centro Jambatu), Linda Trueb, William E. Duellman, John E. Simmons, Rafe Brown (University of Kansas), Alejandro Arteaga, Lucas Bustamante, Jose Vieira (Tropical Herping), John D. Lynch (ICN), James A. Poindexter, Ron Heyer, Addison Wynn (USNM), Darrel Frost, Charles W. Myers, Charles J. Cole, Linda S. Ford (AMNH), Jeff Streicher, David Gower and Mark Wilkinson (BMNH), James Hanken and José Rosado (MCZ), Chris Funk (Colorado State University), Ivan Ineich, Roger Bour, and Jean-Christophe de Massary (MNHN); Janalee Caldwell and Laurie Vitt (OMNH), Santiago Castroviejo-Fisher, Marco Rada (Pontificia Universidad Católica do Rio Grande do Sul), Taran Grant (Universidade de São Paulo), Jane Lyons (Reserva Las Gralarias), Marco M. Reyes (Fundación Óscar Efrén Reyes), Kelly Zamudio (Cornell University), Mario Yánez-Muñoz (INABIO), Brian Kubicki (Costa Rican Amphibian Research Center), Santiago Ron (QCAZ), Ana Almendáriz (EPN), Jean-Marc Touzet and Ana María Velasco (FHGO), Carolina Reyes-Puig, Emilia Peñaherrera and David Brito (ZSFQ), Mauricio Ortega-Andrade (Ikiam) and Andrew J. Crawford (Smithsonian Research Tropical Institute, Panama). For assistance and comments during the development of this project, we extend our sincere gratitude mainly to Luis A. Coloma, Martín R. Bustamante, Elisa Bonaccorso, Santiago Castroviejo-Fisher, and Marco Rada. We also thank Alejandro Arteaga, Lucas Bustamante, Eduardo Toral, Gabriela Gavilanes, Nathalia Valencia, Carolina Reyes-Puig, Diego Páez-Moscoso, Andrea Encalada, Ítalo Tapia, Nicolás Peñafiel, Diana Flores, Gabriela Nicholls, Gene Schupp, Henry Imba, Esteban Suárez, César Barrio-Amorós, Mario Yánez-Muñoz, Jonathan Guillemot, Mark Mulligan, Susana Cárdenas, Jesse Delia, Janeth Lessmann, Jeffrey Arellano, David Brito, Emilia Peñaherrera, Ana Nicole Acosta, Gabriel Muñoz, Ernesto Villacís, Katiuska Valarezo, María Olga Borja, Sebastián Cruz, María Elena Heredia, Andrés León-Reyes, Wendy Loor, Pablo Melo, Carolina Proaño, Daniel Proaño, Geovanna Robayo, Javier Robayo, Mayer Rodríguez, and Tomi Sugahara for their help in numerous ways (discussions, fieldwork, labwork, photography). Several friends and colleagues contributed photographs for this article; their names are listed in the figure captions, but we would like to explicitly thank Luis Coloma, Alejandro Arteaga, Jose Vieira, Lucas Bustamante, Jesse Delia, and Jaime Culebras. Research permits in Ecuador were issued by the Ministerio del Ambiente (#033-IC-FAU-DNBAPVS/MA, #56-IC-FAU/FLO-DPN/MA, #05-2013-IC-FAU-DPAP-MA, MAE-DNB-CM-2015-0017). JMG thanks John D. Lynch, Angela Suárez, Celsa Señaris, Chris Funk, Cameron Ghalambor, and Cesar Barrio-Amorós for their hospitality during visits to Colombia, Venezuela, and United States. DFCH is grateful to George R. Zug, W. Ronald Heyer, Robert P. Reynolds, Kenneth A. Tighe, Steve W. Gotte, Carole C. Baldwin, and Mary Sangrey for their support at the USNM, to Julian Faivovich, Taran Grant, Anita Peñaherrera, Jean-Marc Touzet, Claudia Torres-Gastello, Juana Suarez, Jeff Streicher, and Leonardo Zurita for their support during his visits to New York, Paris, Lima, and London; and to David Romo, Consuelo Barriga de Romo, Kelly Swing, Vlastimil Zak, Francisco Vintimilla, Carlos Burneo, Carolina Proaño, Ana Sevilla, and Roque Sevilla for their support for fieldwork. *Juan Manuel dedicates this study to Nina and Elisa*.

**Conflicts of Interest:** The authors declere no conflict of interest.