The herpetofauna of the Eastern Afromontane: evolutionary history, biogeography and conservation

There is consensus that we are in the midst of the sixth great mass extinction (Barnosky et al., 2011), with a current rate of extinction around 1,000 times the background rate. Current extinction risk has, however, been formally evaluated for less than 5% of the world's described species, but information on the proportion of total species assessed and threatened offers a clear indication of the current trend of biodiversity loss (Baillie et al., 2004). The situation is particularly critical for the class Amphibia, with nearly one-third (32%) of the world’s amphibian species listed as threatened (IUCN, 2014). The Eastern Afromontane region is one of the most diverse areas on the Earth. A large proportion of its biodiversity and underlying evolutionary history is concentrated in small forest fragments on mountains, where both ancient lineages and more recently speciated taxa co-occur and a large proportion of the currently known taxa is formally undescribed. Compared to other tropical areas of the planet, East Africa has been characterized by pronounced climatic and geological turbulence, which, has led to a general faunal impoverishment. Despite this, the combination of the age of the forest fragments, their geographic and ecological isolation and the habitat persistence, makes the vast complex of forest fragments an ancient and stable archipelago of individually evolving sites, especially for the less vagile organisms. Research that assesses the actual patterns of diversity of the region and the definition of the most appropriate conservation strategies that can be effective in a world changing at unprecedented rate is crucial and timely. The aims of this PhD were to investigate historical and current biogeography of the herpetofauna of the Eastern Afromontane, to describe patterns of species richness and endemism across the region and to identify some of the main environmental drivers that have played a significant role in shaping the current scenario. Focused analyses on forest vipers (Atheris) and on dwarf forest bufonids (Nectophrynoides) were carried out in order to understand elements of evolutionary history of the herpetofauna across the region and to prioritize sites for conservation. Chapter 2 focuses on the past and present of herpetological discoveries in the Eastern Afromontane region and identified priority areas where future research could lead to significant herpetofaunal discoveries. I discuss how survey effort, different taxonomic approaches and the use of biodiversity indexes, have important implications for our understanding of the importance of individual sites since both species delimitation methods and the use of indices capture different aspects of biological diversity. The chapter includes 5 appendixes of which three are published papers – two on the description of species new to science and one is a herpetological inventory of a previously overlooked submontane forest in Tanzania. In the fourth appendix I identify priority sites for further inventories (e.g Gura Ferda in Ethiopia, Imatong Mts. in South Sudan, Itombwe and Misotshi-Kabogo ranges in DRC and Mt. Chiperone in Mozambique). The fifth appendix introduces a work-in-progress to publish an e-book on the region’s amphibians and reptiles, which will aid inventory and ecological work across the region. Chapter 3 investigates patterns of species richness and endemism of mountainrestricted amphibians across the Eastern Afromontane region by mapping the distributions of 3,488 geo-referenced records of 274 taxa. Results show a strong geographical congruence between species richness and endemism and how the Albertine Rift and the Eastern Arc mountains appear to have accumulated high amphibian diversity in different ways, with the latter having exceptional diversity despite their small sizes. Finally, results failed to support the idea that the Eastern Afromontane is a discrete zoogeographic region. Chapter 4 focuses on the phylogenetic relationships and historical biogeography of the East African vipers in the genus Atheris and explores temporal and spatial relationships between the different species across Africa. Results showed congruent temporal patterns that link diversification to major tectonic and aridification events within East Africa over the last 15 million years. This points to a diversification pattern of the East African species consistent with a scenario of a delayed direct West-East colonization of the Eastern Arc Mountains, influenced by the formation of the western rift. Possible cryptic taxa in the Atheris genus are also identified. Chapter 5 examined radiations and evolution of forest-associated toad in the genus Nectophyrnoides, through phylogenetic reconstruction, finding that the Eastern Arc Mountains are characterized by the presence of both very ancient paleoendemic species, along with more recent radiations. I then identify key conservation sites using different importance metrics. Congruence between richness of Nectophrynoides and amphibians as a whole was strongly positive, suggesting that this now better-known and conspicuous group could act as a proxy for assessing relative importance of sites for amphibians generally. The high spatial and elevational turnover of Nectophrynoides species indicates the importance of conserving forest at all altitudes and across the entire fragments in order to address the differences that exist between sites at different altitudes, within the same fragment, and at similar altitudes in different fragments on the same mountain block. The results of this thesis highlight the biological importance of the Eastern Afromontane as a key area for the study of the evolution of life and biodiversity conservation, both at African and global levels. It provides original, updated knowledge on species occurrence and biogeographic pattern at regional level. Results also provide and interpretation of the signature left by geographic and climatic events in the pattern of species diversification, clarifying the importance of specific historical events in shaping what we see across the Eastern Afromontane today. The recent surge in biodiversity studies, including the remarkable increase in species description, represents a significant advance in geographic sampling and this, coupled with the effectiveness of new methods for delimiting species, is helping in the assessment of the actual biological value of areas. Furthermore the increasing availability of genetic information on taxa should promote the use of phylogenetic indexes in order to move from a conservation approach solely based on species richness to a more inclusive one, that can inform conservation on the underlying functional diversity and evolutionary potential both at species and site level. The Eastern Afromontane represents the most important area of mainland Africa for conservation of amphibians and reptiles and it offers an extraordinary conservation challenge. Because of the extreme species turnover across mountain ranges and individual forest fragments, a small number of protected areas, however well managed and resourced, will never fully capture the biodiversity of the region. Thus, a specific strategy aimed at identification and implementation of conservation initiatives at forest fragment scale must be considered. Moreover, as a reaction to a lucid assessment of global trends in population growth and associated habitat and species loss and increasing resource demands, there is an urgent need to try new conservation approaches. In the context of a more holistic and radical approach to biodiversity conservation, an active management of the surrounding matrix of the protected areas should be taken into consideration, with the aim of maintaining connectivity between areas of less disturbed habitat and to minimize damage to biodiversity, for unprotected lands, where resource extraction, agriculture, and other productive activities occur

The Nguru mountains of Tanzania, an outstanding hotspot of herpetofaunal diversity

Despite the vicinity of a major road, the rainforests of the South Nguru Mountains in eastern Tanzania were virtually unexplored until 2004, particularly from a herpetological point of view. Several surveys were conducted between 2004 and 2006 with the aim of providing a comprehensive list of the amphibian and reptile species of this overlooked hotspot of biological diversity. The surveys resulted in this assessment of the herpetofaunal diversity, with 92 species recorded, of which 15 represent new records for this area, and the discovery of 16 species new to science, all of which are likely to be strictly endemic to the Nguru Mountains. Pressure on the forests, particularly the lowland forests, remains high. A conservation planning process is now underway that is attempting to address the loss of these critically important forests. These results, documenting the high species richness and the outstanding number of putative endemics of the forests, strongly highlight the biological importance of the South Nguru Mountains and place them among the most important sites for the conservation of herpetofauna in Africa

Species boundaries and biogeography of East African torrent frogs of the genus Petropedetes (Amphibia: Anura: Petropeditidae)

Based on morphology there are currently three described East African species of torrent frogs belonging to the tropical African genus Petropedetes (P. dutoiti, P. martiennseni and P. yakusini). Taxonomic uncertainties are evident within this group: a putative new species reported from the Nguru Mountains and the species P. yakusini have a fragmented distribution across the Southern Tanzanian highlands. We conducted a molecular systematic study, sampling populations of East African petropedetids occurring in the mountains of Tanzania (P. martiennseni and P. yakusini), to investigate species boundaries and biogeography. Data provide evidence for the recognition of well-defined genetically distinct populations occurring in each mountain fragment (for P. yakusini), and further support for a new undescribed species from the Nguru Mountains. We examine phylogenetic patterns in relation to the biogeographic history of the region

Estimating abundance and habitat suitability in a micro-endemic snake: the Walser viper

Recently described species suffer lack of information that hampers setting up appropriate conservation strategies. The situation is particularly complex with micro-endemic snakes, for which detection and monitoring are particularly challenging. The Walser viper Vipera walser is a recently described snake inhabiting a small area of the SW Italian alps. We combined information on species distribution with repeated monitoring to identify the areas most suitable for the species, and to obtain estimates of species abundance. Species distribution models were used to identify the topographical, climatic, and land-cover features related to the occurrence of vipers. Furthermore, repeated transects and N-mixture models were used to estimate abundance and to identify factors related to the variation of abundance. The available data suggested that the species has a disjunct range, with a Northern range of ~45 km2 , and a southern range of ~225 km2. Distribution models suggested that vipers are associated with areas with open egetation,altitude between 1300 and 2300 m, high precipitation, low forest cover, low slope, and southern aspect. N-mixture models confirmed very low detection probability of these vipers, and suggested that the species has a low abundance,with the highest abundance in south-facing plots. We provide the first quantitative information on habitats and abundance variation for Walser vipers. The broad confidence intervals of abundance estimates exemplify the complexity of providing range-wide measures of abundance for secretive species. Given the narrow range of these vipers, continuous monitoring is required to understand how they respond to ongoing environmental changes in mountainous areas

Intradural Extramedullary Plasmacytoma in a Dog

Background: Plasmacytoma is a neoplasm originating in plasma cells, derived from B lymphocytes. Extramedullary presentation is the most common form of plasmacytoma, mainly in the skin and rarely in other tissues, including the vertebral canal. Extramedullary plasmacytoma (EMP) occurs more frequently in senile dogs and rarely in cats and some dog breeds have predisposition for this type of neoplasm. The aim of this study was to report plasmacytoma in a dog located adjacent to the thoracic vertebrae.Case: An approximately 5-year-old mongrel male with sudden paraplegia resulting from upper motor neuron injury was admitted to a University Hospital. The dog presented a six-centimeter diameter mass in the dorsal region, adjacent to the thoracic vertebrae. The leucogram presented mild leukopenia by eosinopenia and lymphopenia. Myelogram associated with epidurography showed a fill failure in the contrast column between the seventh and ninth thoracic vertebrae, and there were no osteolysis points in the thoracic vertebrae, adjacent to the neoplastic mass. The evaluation of the intervertebral spaces between the fourth to ninth thoracic vertebrae presented no increase in radiopacity, nor alterations in the size of intervertebral spaces, indicating intervertebral disc extrusion. The laterolateral radiographs showed an alignment of the vertebrae and vertebral canal, without presence of bone neoformations on the ventral face of the vertebral bodies. The animal was euthanized and fragments of mass were collected for histological analysis. Macroscopic findings presented a non-encapsulated well-delimited mass, with color ranging from white to reddish. Microscopically, there was proliferation of moderately differentiated round cells in bone and muscle tissues, arranged in mantle, with moderately eosinophilic cytoplasm, and rounded nucleus with eccentric location, coarse chromatin sometimes indistinct. These cells presented moderate anisocytosis and, in general, one mitosis per field. Intradural extramedullary plasmacytoma was diagnosed.Discussion: The present report presented a diagnosis of intradural EMP, generating compression of the spinal cord because of its invasive growth, reaching the canal and, thus, reducing its lumen. The clinical status of the dog was compatible with thoracolumbar lesion of upper motor neuron, which causes paresis and ataxia in pelvic limbs. In this case, it was not possible to establish early diagnosis because the dog had been rescued from the streets recently. Radiographic examination was important to identify the real size of the mass, and the myelography confirmed spinal cord compression. The definitive diagnosis was obtained by histological examination. Although extramedullary plasmacytoma is a low-frequency neoplasm, in the present report, its manifestation was atypical, more aggressive, invading the medullary canal and compressing the spinal cord. Spinal cord compression may lead to degeneration of axons because of alterations in the vascular permeability associated with local inflammatory process, reducing the oxygen supply to nervous tissues, resulting in cellular degeneration. The occurrence of atypical neoplastic growth should be included as a differential diagnosis for lesions in upper motor neuron in cases of spinal cord compression. This is an atypical location for this kind of neoplasia, and cases of medullary compression of neoplastic masses should be included as a differential diagnosis

Exploration into the hidden world of Mozambique’s sky island forests:new discoveries of reptiles and amphibians

We carried out a survey of reptiles and amphibians within Afromontane forest and woodland slopes of three inselbergs in northern Mozambique (Mount Mabu, Mount Namuli, and Mount Ribáuè). A total of 56 species (22 amphibians and 34 reptiles) were recorded during the current survey. Our findings substantially increase the number of herpetofaunal species recorded from these mountains (Mount Ribáuè 59%, Mount Mabu 37%, and Mount Namuli 11% of the total species), including one new country record and several putative new species. An updated checklist of the herpetofauna of these mountains is presented

Long-term data for endemic frog genera reveal potential conservation crisis in the Bale Mountains, Ethiopia

Populations of many frogs have declined alarmingly in recent years, placing nearly one third of the \u3e 6,000 species under threat of extinction. Declines have been attributed largely to habitat loss, environmental degradation and/or infectious diseases such as chytridiomycosis. Many frogs undergo dramatic natural population fluctuations such that long-term data are required to determine population trends without undue influence of stochastic factors. We present long-term quantitative data (individuals encountered per person hour of searching) for four monotypic frog genera endemic to an Afromontane region of exceptional importance but growing conservation concern: one endemic to the Ethiopian highlands (Spinophrynoides osgoodi) and three endemic to the Bale Mountains (Altiphrynoides malcolmi, Balebreviceps hillmani, Ericabatrachus baleensis), collected during 15 field trips to the Bale Mountains between 1971 and 2009. Only a single confirmed sighting of S. osgoodi has been made since 1995. The other three species have also declined, at least locally. E. baleensis appears to have been extirpated at its type locality and at the same site B. hillmani has declined. These declines are in association with substantial habitat degradation caused by a growing human population. Chytrid fungus has been found on several frog species in Bale, although no dead or moribund frogs have been encountered. These results expose an urgent need for more amphibian surveys in the Bale Mountains. Additionally, we argue that detrimental human exploitation must be halted immediately in at least some parts of the Harenna Forest if a conservation crisis is to be averted

No ecological opportunity signal on a continental scale?:Diversification and life-history evolution of african true toads (Anura: Bufonidae)

The niche-filling process predicted by the “ecological opportunity” (EO) model is an often-invoked mechanism for generating exceptional diversity in island colonizers. Whether the same process governs lineage accumulation and trait disparity during continental colonization events is less clear. Here, we test this prediction by investigating the rate dynamics and trait evolution of one of Africa's most widespread amphibian colonizers, the true toads (Bufonidae). By reconstructing the most complete molecular phylogeny of African Bufonidae to date, we find that the diversification of lineages in Africa best conforms to a constant rate model throughout time and across subclades, with little support for EO. Evolutionary rates of life-history traits have similarly been constant over time. However, an analysis of generalists and specialists showed a shift toward higher speciation rates associated with habitat specialization. The overall lack of EO signal can be interpreted in a number of ways and we propose several explanations. Firstly, methodological issues might preclude the detection of EO. Secondly, colonizers might not experience true EO conditions and due to the size, ecological heterogeneity and age of landmasses, the diversification processes might be more complex. Thirdly, lower speciation rates of habitat generalists may have affected overall proliferation of lineages

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions

High Prevalence of the Amphibian Chytrid Fungus (\u3cem\u3eBatrachochytrium dendrobatidis\u3c/em\u3e) across Multiple Taxa and Localities in the Highlands of Ethiopia

Surveys of the potentially lethal amphibian chytrid fungus (Batrachochytrium dendrobatidis - Bd) in Africa are patchy, especially in some regions of high species endemicity. We present results of the first Bd surveys of wild amphibians in Ethiopia, for two upland regions on either side of the Rift Valley: the Bale Mountains and the Kaffa region. Surveys were opportunistic so that robust interpretation of the data is limited. Utilizing diagnostic qPCR assays, 51 out of 120 frogs (14 species in 10 genera) tested positive for Bd at altitudes of 1,620–3,225 m, across all genera and species, and all but two localities. Prevalence was not significantly different between the two regions or two years (2008, 2009) sampled. Prevalence and parasite load was higher in species with aquatic tadpoles than those with terrestrial early life-history stages, but these differences were not significant. Impacts of Bd infection were not investigated, but no dead or dying frogs were found. This is the first report of Bd in Ethiopia, a country in which approximately 40% of its more than 60 species are endemic. Declines have occurred in some frog species in some localities in Ethiopia, and although habitat degradation is a likely cause in at least some places, further studies of Bd in Ethiopia are required to understand if it is a threat