Discovery of two new populations of the rare endemic freshwater crab Louisea yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019 (Brachyura: Potamonautidae) from the Ebo Forest near Yabassi in Cameroon, Central Af - rica, with recommendations for conservation action

: The endemic freshwater crab, Louisea yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019, is currently only known from three populations in the biodiversity-rich rainforests of southwestern Cameroon. The first record of L . yabassi dates back to 1908 from Yabassi, while the other two populations were discovered in December 2019 and March 2020 from the Ebo Forest near Yabassi. These specimens were initially identified as L . edeaensis (Bott, 1969), but were subsequently assigned to L . yabassi . The newly-discovered populations of L . yabassi provided important data on its habitat, population structure and geographical distribution, all critical knowledge for conservation measures. Reported here are the anthropogenic threats to L . yabassi and its rainforest habitat, which include forest destruction, agricultural encroachment, water pollution and firewood collection. This information is inherently useful in the assessment of the extinction risk of L . yabassi and highlights the importance of implementing strategies for preserving primary rainforest and its associated aquatic habitats in Central Africa.


INTRODUCTION
The present work arises from the discovery of two new populations of Louisea yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019, from the Ebo Forest near Yabassi in southwestern Cameroon. This rare endemic species of freshwater crab is currently known from just three small populations found in Yabassi (collected in 1908;Cumberlidge 1994bCumberlidge , 1999, and the Ebo Forest (collected in December 2019 andMarch 2020;Mvogo Ndongo et al. 2019).
The Ebo Forest, where L. yabassi was rediscovered ( Figure 1, Image 2), is the largest remaining tract of primary lowland and submontane rainforest in this part of Africa, and is drained by the Wouri and Dibamba rivers that flow into the Atlantic Ocean. This forested area represents a key biodiversity hotspot in southwestern Cameroon for a number of freshwater taxa including crabs. The freshwater catchments of the Ebo Forest are important spawning grounds for fish and invertebrates, and the forest is also a refuge for charismatic wildlife including monkeys, chimpanzees, gorillas, manatees, elephants, birds, turtles, snakes, and amphibians (Cumberlidge 1994b(Cumberlidge , 1999Morgan & Abwe 2006;Morgan et al. 2013).
The rediscovery of L. yabassi after over 110 years means that living specimens of this species are now available for scientific studies that allow for DNA analysis, a description of its habitat, and ecological and population studies. The difficult taxonomy and the chronic lack of material in the past meant that the original specimens from Yabassi were initially identified as L. edeaensis (Bott, 1969) by Cumberlidge (1994a, 1999) and Mvogo Ndongo et al. (2017a. Preliminary surveys indicated that this newlyrediscovered species is facing immediate threats to its freshwater habitat from forest destruction, agricultural encroachment, water pollution, and firewood collection. Presented here is the necessary field data for an IUCN Red List extinction risk assessment of this species including its specific habitat requirements, population trends, distribution, and threats (Mvogo Ndongo et al. 2017a). The importance of assessing the extinction risk of L. yabassi is underlined by the status of two other rare endemic species assigned to Louisea in southwestern Cameroon, L. edeaensis (Cumberlidge, 1994) and L. balssi (Bott, 1969) that are both currently listed as Endangered species (EN) on the International Union for Conservation of Nature (IUCN) Red List.
The semi-terrestrial species of freshwater crabs endemic to the rainforests of southwestern Cameroon are of great conservation importance and are also the most threatened by anthropogenic activities in these forests (see Table 1). These species are: Buea asylos (Cumberlidge, 1993), B. bangem Mvogo Ndongo, von Rintelen, Tomedi-Tabi & Cumberlidge, 2020, B. mundemba Mvogo Ndongo, von Rintelen & Cumberlidge, 2020, B. nlonako Mvogo Ndongo, von Rintelen & Cumberlidge, 2020, Louisea balssi (Bott, 1969, L. edeaensis (Bott, 1969), L. nkongsamba Mvogo Ndongo, von Rintelen & Cumberlidge, 2019, L. yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019, and Sudanonautes tiko Mvogo Ndongo, Schubart & Cumberlidge, 2017. Two of these species, L. balssi and L. edeaensis, are already assessed as EN by the IUCN Red List, and were previously thought to be extinct until their rediscovery in 2017and 2018(IUCN 2003Cumberlidge 2008a,b;Cumberlidge et al. 2009;Mvogo Ndongo et al. 2017a;2018). These threatened species of freshwater crabs are found in an area of great conservation interest, because their aquatic habitats also serve as key spawning grounds for fish and invertebrates, as well as refugia for other forest wildlife, e.g., monkeys, drills, chimpanzees, and gorillas as well as manatees, elephants, birds, turtles, snakes and amphibians (Cumberlidge et al. 2019;Mvogo Ndongo et al. 2017a-c, 2018, 2019, 2020. Consequently, these areas increasingly attract scientists and tourists. The high rate of endemism of freshwater crabs in southwestern Cameroon reflects their low dispersal abilities arising from a lifecycle that includes direct development of eggs into young crabs, without the highly dispersive planktonic larval stages seen in marine crabs (Cumberlidge & Daniels 2007). Most of these endemic species of freshwater crabs (B. bangem, B. mundemba, L. balssi, and L. edeaensis) are still only known from J TT a single population. Other species are better known and are represented by at least two populations, most likely revealing strong phylogeographical structuring. Despite advances in our knowledge of the taxonomy, habitat, and distributional range of each of these species, information on their reproductive biology, ecology, phylogeography, and evolutionary relationships is still extremely limited. This is a problem because of the urgent need to provide vital biological data that are necessary for the management of the freshwater crabs of southwestern Cameroon and to monitor changes in their populations and habitat as a result of conservation interventions. The present study aims to formulate guidelines towards a more sustainable use of forest and aquatic resources from southwestern Cameroon, and lay the groundwork for a conservation assessment of those species of endemic freshwater crabs most vulnerable to anthropogenic threats.

MATERIAL AND METHODS
The data presented here for L. yabassi were compiled during field surveys of the rivers, streams, wetlands, and nearby land in the Ebo Forest near Yabassi in December 2019 and March 2020. The number of plants destroyed by natural and human activities was assessed at the locality and around the sampling sites. The pH and temperature of water samples where crabs occurred were measured (Table 2), and specimens were identified using the keys provided by Cumberlidge et al. (2019) and Mvogo Ndongo et al. (2019;2020). Carapace measurements of each specimen were taken using digital callipers, and sex and age were recorded. Photographs of freshly caught specimens and the habitat from where they were collected (Image 1) are also provided. Most specimens were returned to their place of origin, except for one adult male and female, which were preserved in ethanol for subsequent detailed morphological and molecular analyses.

RESULTS
The updated distributional range of L. yabassi is presented in Figure 1A. The two populations recently discovered in the Ebo Forest were collected from two small streams ('stream-1' and 'stream-2') that flow independently into the Dibamba River at the locality N'dogbanguengue ( Figure 1B). The first population (15 males and five females) was found ca. 10m from the middle of stream-1 in December 2019 ( Figure 1B, Image 2A). At that time of the year, the water levels of the streams and rivers are at their peak and are difficult to sample, so all of our specimens of L. yabassi were collected from semi-aquatic habitats on land, adjacent to the streams in damp conditions in puddles, under fallen leaves, and in burrows. The second population of L. yabassi (nine males and six females) was found ca. 8m from the middle of stream-2 in March 2020 ( Figure 1B, Image 2B). At this time of the year, the water levels of the streams and rivers are much lower, so that sampling in the large streams was now possible under the rocks and boulders that sheltered crabs (Image 2). Most of the natural vegetation at these localities had been destroyed as a result of human activities (by foresters, farmers, and others). The trees had been logged to build huts, camps, and toilets. The remaining vegetation in these locations had been removed for intensive agricultural practices and for firewood. In addition, the farmers encroaching on these habitats use agro-chemicals and pesticides on their crops, and these pollutants eventually drain into the aquatic systems and can poison the freshwater communities.

Current threats to Louisea yabassi
Deforestation, together with intensive and encroaching agricultural practices, are serious issues. These activities present imminent threats to southwestern Cameroon's rich aquatic biodiversity, which is of concern, because this area includes a number of rare endemic species. Large scale habitat disturbance also has a negative impact on the culture of the indigenous people of this region, who depend on the intact forest for their livelihoods. For example, the Pygmy forest people of this part of Cameroon express their problems as follows. " […] We are in the midst of huge desolation, we no longer recognize the forest, we no longer understand what is happening. Our forests change from one day to the next. What future awaits our children? The settlements destroy the forest, and the felling of trees prevents us from gathering honey to feed our children. The noise

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Deforestation has direct effects on the aquatic environment and indirect impacts from changes within the drainage basin, both of which will affect rare endemic forest species, such as L. yabassi, that depend on the forest canopy remaining closed and intact (Cumberlidge & Sachs 1991;Dudgeon et al. 2005;Mvogo Ndongo et al. 2018). Not only does deforestation expose aquatic systems and their inhabitants to the heating and drying effects of direct sunlight, but local farming practices also release pollutants such as agro-chemicals potentially affecting the eggs, hatchling-carrying females, and adults of L. yabassi. In addition, clearing the tropical rainforest leads to increased agricultural encroachment and firewood collection, which further impacts the habitat of L. yabassi.

Conservation recommendations
The extinction risk status of L. yabassi has not yet been assessed, but the data now available allow us to present a preliminary pre-assessment. Unfortunately, the two additional localities for this species are so close together that IUCN Red List protocols treat them as a single location. This is because the calculation of the extent of occurrence (EOO, the area contained within the minimum convex polygon around all sites of present occurrence) and the area of occupancy (AOO, the area within the EOO that is actually occupied by the taxon) requires at least three locations. Despite this, if an IUCN Red List extinction risk assessment were to be made, then we anticipate that L. yabassi would be assessed as Critically Endangered (CR) under criterion B alone using the small number of locations, the low population levels, the disruption of the habitat, and the severity of the immediate known threats.
The obvious nature and the large scale of the threats to the habitat of L. yabassi in the Ebo Forest mean that a conservation action plan needs to be formulated if this threatened species is to be protected from the anthropogenic threats it is facing. Conservation actions include the monitoring of populations, studies of population genetics, and protection against threats. These actions would also include a strategy for communication and education of the stakeholders in the local community about the consequences of engaging in forest destruction, firewood collection, and agricultural encroachment. Local knowledge from indigenous people should also form part of the conservation messaging. Conservation action on a local scale is feasible because of its 'low-tech' approach that is necessary (and successful) in the context of rural Africa. The content of the educational messaging needs to be structured to highlight the need for protection and to emphasise the potential advantages brought to the area by increased numbers of visitors (eco-tourists and scientists). The targeted stakeholders in the local communities should be brought to understand that L. yabassi is found only in the remaining rainforest tracts around Yabassi, and that this habitat is globally unique. The semi-terrestrial lifestyle of L. yabassi means that its habitat ranges from permanent streams to seasonal shallow waters and wetlands, all of which are impacted by intensive destructive agricultural practices that degrade and pollute the natural vegetation. The release of pesticides into the environment containing substances that either harm or kill most species of invertebrates and vertebrates (most certainly including L. yabassi) needs to be mitigated. In addition, these pesticides have been linked to a wide range of human health hazards ranging from headaches and nausea to cancer, reproductive harm, and endocrine disruption. Pesticides have also been linked to nerve, skin, and eye irritation, dizziness, fatigue, and even sometimes fatal systemic poisoning (Roberts & Reigart 2013).
In addition to highlighting the problems facing the rainforest around Yabassi and the Ebo Forest, the education of the local people and the Government includes the need for awareness building about the

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broader lasting benefits of conservation action. These include hosting and guiding tourists and scientists from Cameroon and all around the world attracted by Africa's intact tropical rainforest ecosystems and associated unique wildlife. Further, the local markets and hospitality industry in the area will benefit from the increased flow of visitors, and this will contribute to the development of the local community. The problem of firewood collection requires the promotion of the legal harvesting of forest resources found in the forested habitat of L. yabassi. This distinction is important because there are no alternative sources of firewood other than the natural rainforest itself. Legal exploitation of forest resources involves avoiding cutting young trees and bushes, and only cutting off dead branches or using parts of the trees that fall naturally. It also means that the vegetation that falls around streams should be left untouched, because this constitutes good habitat for aquatic invertebrates, including L. yabassi. As for agricultural encroachment, it is necessary to recommend to farmers to focus their attention on land that has already been used for cultivation and direct them away from the natural vegetation around the streams that form the habitat of L. yabassi. What this study suggests is that farmers should curtail their activities, avoid disturbing the natural vegetation near aquatic habitats and keep these watersheds free from pesticides.
The success of this conservation action also requires public training/education sessions (workshops) aimed at involving a wider section of the local community beyond those individuals encountered during field work. Monitoring strategies for L. yabassi mean that local young people, students, and engineers need to be trained in how to communicate the above educational messages, and how to undertake routine monitoring to collect data on the organisms present in a habitat and general ecosystem health. The local authorities (e.g., the chiefs of the villages, sub-prefets, and prefets) need to be directly involved, because educational messaging in the community is an ongoing process, and the involvement of community leaders is key to engaging the wider community in these conservation efforts. The targets of the educational messages (local people, fishermen, farmers, foresters, hunters, and other scientists) should also be made aware of the need to constantly monitor the health of the habitat, such as monitoring changes in forest cover, agricultural practices, and the expansion of farms. As conservationists it is of great importance to inform people on the broader role of biodiversity (vertebrates and invertebrates) in the rainforest ecosystem and why illegal poaching of wildlife (e.g., gorillas, chimpanzees, and numerous bird species) and unsustainable fishing practices (such as small mesh size of nets, unenforced fishing seasons, and fishing with poison) requires controlling.

Threatened Taxa
Author details: Pierre A. Mvogo Ndongo, Ph.D in Biology (speciality, Zoology) is interested on taxonomics/systematics, phylogenetics and conservation of West and Central African freshwater and mangrove Decapod Crustaceans, as well as sustainable management of the ecosystems they depend from. He is the Chief of Research unit for taxonomics/systematics, production and sustainable management of aquatic animals at the Department of Management of Aquatic Ecosystems, Institute of Fisheries and Aquatic Science, University of Douala, Cameroon. Thomas von Rintelen is an evolutionary biologist with a focus on molecular phylogenetics and genomics at the Museum für Naturkunde, Berlin, Germany. He is strongly interested in the diversity and biogeography of tropical freshwater invertebrates and their conservation. Much of his research is focused on SE Asia, but he is also active in Cameroon. Christoph D. Schubart works as an Assistant Professor in the Department of Zoology and Evolutionary Biology at the University of Regensburg in Germany. investigates the evolution and diversification of aquatic arthropods (mainly Crustacea). With his study group he investigates the diversification and speciation history of aquatic arthropods, mainly decapod Crustacea from marine and freshwater environments. Paul F. Clark is a decapod crustacean specialist at The Natural History Museum, London, UK who does research on brachyuran crabs and their larvae. He has a strong interest in non-native UK species, and he is currently studying co-endemicity of paragonimiasis with respect to its links with Sub-Saharan African species of freshwater crabs. Kristina von Rintelen is a researcher at the Museum für Naturkunde, Berlin, Germany, specialized on crustaceans, especially freshwater shrimps. She is particularly interested in the taxonomy and speciation of tropical species as well as the implementation of integrative taxonomy approaches. Kristina has also been involved in Red List assessment of freshwater crustaceans. Alain Didier Missoup is an Associate Professor in the Zoology Unit of the Laboratory of Biology and Physiology of Animal Organisms in Faculty of Science, University of Douala. He studies diversification patterns promoting the biodiversity in tropical Africa and bushmeat trafficking in Central Africa. He currently works on the fauna of Ebo Forest where the species Louisea yabassi was rediscovered. Christian Albrecht is an Associate Professor of Biogeography at Justus Liebig University Giessen, Germany. He is interested in biodiversity, biogeography, evolution, and conservation of invertebrates in African freshwater, focusing mostly on molluscs and crustaceans. Muriel Rabone is a sample coordinator for SCAN (Schistosomiasis Collection at the Natural History Museum) in London, UK. She is currently studying the coendemicity of paragonimiasis and tuberculosis in Sub-Saharan Africa and she is utilising existing data to improve public health outcomes. T. Efole Ewoukem is an Associate professor working on fishes production and conservation of aquatic plants from Cameroon. Joseph L. Tamesse is a Professor of Biology working on taxonomics/systematics and conservation of Central African Invertebrates. Minette Tomedi-Tabi Eyango, is a Professor of Aquaculture, with interest on production and aquatic bio-resources from Southern-Cameroon. She is the active Director of the Institute of Fisheries and Aquatic Science, University of Douala at Yabassi, near Eboforest, Cameroon. Neil Cumberlidge is a Professor of Biology who conducts research on African freshwater crab conservation, biogeography, systematics and evolution and is the Chair of the Freshwater Crustacean Specialist Group of the International Union for the Conservation of Nature. He is an active conservationist who led the global Red Listing of the freshwater crabs of the world.
Author contributions: Pierre A. Mvogo Ndongo collected the data, Pierre A. Mvogo Ndongo, Neil Cumberlidge, Thomas von Rintelen, Christoph Schubart, Paul Clark, Christian Albrecht, and Muriel Rabone analysed the data and wrote the manuscript. Kristina von Rintelen, Alain Didier Missoup, Thomas Efole Ewoukem, Joseph L. Tamesse, and Minette Tomedi Tabi-Eyango gave valuable inputs in data processing and made constructive improvements to the manuscript. www.threatenedtaxa.org The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.

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