Relicts in the mist: Two new frog families, genera and species highlight the role of Pantepui as a biodiversity museum throughout the Cenozoic.

The iconic mountains of the Pantepui biogeographical region host many early-diverging endemic animal and plant lineages, concurring with Conan Doyle's novel about an ancient "Lost World". While this is the case of several frog lineages, others appear to have more recent origins, adding to the controversy around the diversification processes in this region. Due to its remoteness, Pantepui is challenging for biological surveys, and only a glimpse of its biodiversity has been described, which hampers comprehensive evolutionary studies in many groups. During a recent expedition to the Neblina massif on the Brazil-Venezuela border, we sampled two new frog species that could not be assigned to any known genus. Here, we perform phylogenetic analyses of mitogenomic and nuclear loci to infer the evolutionary relationships of the new taxa and support their description. We find that both species represent single lineages deeply nested within Brachycephaloidea, a major Neotropical clade of direct-developing frogs. Both species diverged >45 Ma from their closest relatives: the first is sister to all other Brachycephaloidea except for Ceuthomantis, another Pantepui endemic, and the second is sister to Brachycephalidae, endemic to the Brazilian Atlantic Forest. In addition to these considerable phylogenetic and biogeographic divergences, external morphology and osteological features support the proposition of two new family and genus-level taxa to accommodate these new branches of the amphibian tree of life. These findings add to other recently described ancient vertebrate lineages from the Neblina massif, providing a bewildering reminder that our perception of the Pantepui's biodiversity remains vastly incomplete. It also provides insights into how these mountains acted as "museums" during the diversification of Brachycephaloidea and of Neotropical biotas more broadly, in line with the influential "Plateau theory". Finally, these discoveries point at the yet unknown branches of the tree of life that may go extinct, due to global climate change and zoonotic diseases, before we even learn about their existence, amphibians living at higher elevations being particularly at risk.

DNA barcoding of the National Museum of Natural History reptile tissue holdings raises concerns about the use of natural history collections and the responsibilities of scientists in the molecular age.

Natural history collections are essential to a wide variety of studies in biology because they maintain large collections of specimens and associated data, including genetic material (e.g., tissues) for DNA sequence data, yet they are currently under-funded and collection staff have high workloads. With the advent of aggregate databases and advances in sequencing technologies, there is an increased demand on collection staff for access to tissue samples and associated data. Scientists are rapidly developing large DNA barcode libraries, DNA sequences of specific genes for species across the tree of life, in order to document and conserve biodiversity. In doing so, mistakes are made. For instance, inconsistent taxonomic information is commonly taken from different lending institutions and deposited in data repositories, such as the Barcode of Life Database (BOLD) and GenBank, despite explicit disclaimers regarding the need for taxonomic verification by the lending institutions. Such errors can have profound effects on subsequent research based on these mis-labelled sequences in data repositories. Here, we present the production of a large DNA barcode library of reptiles from the National Museum of Natural History tissue holdings. The library contains 2,758 sequences (2,205 COI and 553 16S) from 2260 specimens (four crocodilians, 37 turtles, and 2,219 lizards, including snakes), representing 583 named species, from 52 countries. In generating this library, we noticed several common mistakes made by scientists depositing DNA barcode data in public repositories (e.g., BOLD and GenBank). Our goal is to raise awareness of these concerns and offer advice to avoid such mistakes in the future to maintain accurate DNA barcode libraries to properly document Earth's biodiversity.

A global catalog of primary reptile type specimens.

We present information on primary type specimens for 13,282 species and subspecies of reptiles compiled in the Reptile Database, that is, holotypes, neotypes, lectotypes, and syntypes. These represent 99.4% of all 13,361 currently recognized taxa (11,050 species and 2311 subspecies). Type specimens of 653 taxa (4.9%) are either lost or not located, were never designated, or we did not find any information about them. 51 species are based on iconotypes. To map all types to physical collections we have consolidated all synonymous and ambiguous collection acronyms into an unambiguous list of 364 collections holding these primary types. The 10 largest collections possess more than 50% of all (primary) reptile types, the 36 largest collections possess more than 10,000 types and the largest 73 collections possess over 90% of all types. Of the 364 collections, 107 hold type specimens of only 1 species or subspecies. Dozens of types are still in private collections. In order to increase their utility, we recommend that the description of type specimens be supplemented with data from high-resolution images and CT-scans, and clear links to tissue samples and DNA sequence data (when available). We request members of the herpetological community provide us with any missing type information to complete the list.

How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation.

Development projects in west Central Africa are proceeding at an unprecedented rate, often with little concern for their effects on biodiversity. In an attempt to better understand potential impacts of a road development project on the anuran amphibian community, we conducted a biodiversity assessment employing multiple methodologies (visual encounter transects, auditory surveys, leaf litter plots and pitfall traps) to inventory species prior to construction of a new road within the buffer zone of Moukalaba-Doudou National Park, Gabon. Because of difficulties in morphological identification and taxonomic uncertainty of amphibian species observed in the area, we integrated a DNA barcoding analysis into the project to improve the overall quality and accuracy of the species inventory. Based on morphology alone, 48 species were recognized in the field and voucher specimens of each were collected. We used tissue samples from specimens collected at our field site, material available from amphibians collected in other parts of Gabon and the Republic of Congo to initiate a DNA barcode library for west Central African amphibians. We then compared our sequences with material in GenBank for the genera recorded at the study site to assist in identifications. The resulting COI and 16S barcode library allowed us to update the number of species documented at the study site to 28, thereby providing a more accurate assessment of diversity and distributions. We caution that because sequence data maintained in GenBank are often poorly curated by the original submitters and cannot be amended by third-parties, these data have limited utility for identification purposes. Nevertheless, the use of DNA barcoding is likely to benefit biodiversity inventories and long-term monitoring, particularly for taxa that can be difficult to identify based on morphology alone; likewise, inventory and monitoring programs can contribute invaluable data to the DNA barcode library and the taxonomy of complex groups. Our methods provide an example of how non-taxonomists and parataxonomists working in understudied parts of the world with limited geographic sampling and comparative morphological material can use DNA barcoding and publicly available sequence data (GenBank) to rapidly identify the number of species and assign tentative names to aid in urgent conservation management actions and contribute to taxonomic resolution.

Unexpected rarity of the pathogen Batrachochytrium dendrobatidis in Appalachian Plethodon Salamanders: 1957-2011.

Widespread population declines in terrestrial Plethodon salamanders occurred by the 1980s throughout the Appalachian Mountains, the center of global salamander diversity, with no evident recovery. We tested the hypothesis that the historic introduction and spread of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) into the eastern US was followed by Plethodon population declines. We expected to detect elevated prevalence of Bd prior to population declines as observed for Central American plethodontids. We tested 1,498 Plethodon salamanders of 12 species (892 museum specimens, 606 wild individuals) for the presence of Bd, and tested 94 of those for Batrachochytrium salamandrivorans (Bs) and for ranavirus. Field samples were collected in 2011 from 48 field sites across a 767 km transect. Historic samples from museum specimens were collected at five sites with the greatest number and longest duration of collection (1957-987), four of which were sampled in the field in 2011. None of the museum specimens were positive for Bd, but four P. cinereus from field surveys were positive. The overall Bd prevalence from 1957-2011 for 12 Plethodon species sampled across a 757 km transect was 0.2% (95% CI 0.1-0.7%). All 94 samples were negative for Bs and ranavirus. We conclude that known amphibian pathogens are unlikely causes for declines in these Plethodon populations. Furthermore, these exceptionally low levels of Bd, in a region known to harbor Bd, may indicate that Plethodon specific traits limit Bd infection.

Fire adaptation in Neblinaria celiae (Theaceae), a high-elevation rosette shrub endemic to a wet equatorial tepui.

Neblinaria celiae (Theaceae), a rosette shrub endemic to the exceedingly rainy summit of remote Cerro de la Neblina in southern Venezuela, has a previously undescribed set of adaptations to fire. Its growth form entails sparse branching, massive terminal leaf rosettes, and thick bark. It is highly fire-tolerant, with a survival rate of 93% in a stand recently ignited by lightning, vs. 0% in seven co-occurring woody species. Survival increases sharply with rosette height, favoring a sparsely branched habit that would maximize the rate of upward growth through the sparse fuel layer supported by a sterile substrate. Thick bark and massive rosettes help protect cambial and foliar meristems from brief exposure to high temperatures. Rosettes on shorter plants are exposed to greater damage from fire near the ground and, as expected, are bigger and impound more rainwater; the greater number of leaves nearly balances the greater leaf mortality caused by fire. We relate Neblinaria's growth form to its dominance atop Neblina, to a general model for the evolution of sparse branching, and to the evolution of growth form in other tepui plants.