Two decades ago, the new siluriform family Lacantuniidae was erected to accommodate its sole known extant representative, Lacantunia enigmatica, a morphologically aberrant catfish restricted to the Middle Usumacinta River basin along the Guatemala-Mexico border. While its discovery was unexpected, its proposed phylogenetic placement - nested within a large clade of exclusively African-endemic families rather than closely related to regionally sympatric North American or Neotropical lineages - was baffling. To test this counterintuitive phylogenetic and biogeographic hypothesis, we collected new specimens of L. enigmatica and sequenced, assembled, and annotated for the first time its complete mitochondrial genome. We then constructed the most taxonomically comprehensive mitochondrial data matrix of catfishes to date by including published mitochondrial genomes representing the majority of siluriform families. Using Bayesian co-estimation of phylogeny and divergence times, we inferred the evolutionary position of the puzzling L. enigmatica within a time-scaled global catfish phylogeny. Our results offer improved resolution and understanding of higher-level siluriform relationships and refine the timescale of catfish evolution. Crucially, our findings corroborate the hypothesis that L. enigmatica is sister to the African family Claroteidae and represents a relict lineage that originated in Africa during the Late Cretaceous (74.99 Ma; 95% HPD = 68.58-81.39) and eventually culminated in Middle America. Our results therefore uphold the necessity of transoceanic dispersal - as opposed to Gondwanan vicariance - to explain this otherwise puzzling biogeographic pattern.

Environmental DNA (eDNA) metabarcoding is an emerging non-invasive tool for biodiversity monitoring, but its application to airborne sources remains underexplored, particularly for insects. Moths play key ecological roles as pollinators, prey, and indicators of environmental change, making them valuable targets for monitoring. In this study, we evaluate the effectiveness of airborne eDNA metabarcoding for monitoring Lepidopteran biodiversity, comparing its performance against traditional light-trapping methods. Air samples were collected using compact air filters that were paired with light traps over a 30-day period at two suburban garden sites in the UK. The results from this study were mixed. Both methods identified the same dominant families at similar proportions, but species-level overlap was low across sites (23.7%-32.6%). While eDNA metabarcoding detected 41% fewer species than the light traps, it nevertheless detected species not captured by them. Despite successfully identifying a range of moth species, eDNA read counts did not correlate with moth abundance in the light traps, limiting its usefulness for estimating relative abundance. With increased sampling effort and optimized laboratory protocols, airborne eDNA could become a valuable complementary tool to traditional monitoring methods. However, light traps remain essential for collecting ecological data, such as abundance, body size, and phenological patterns. Integrating eDNA with traditional methods may offer a more comprehensive understanding of moth biodiversity and ecological dynamics. Further work is needed to address methodological limitations, particularly sampling strategies and primer design, before airborne DNA can be reliably applied in biodiversity monitoring.

Many island archipelagos sit on shallow continental shelves, and during the Pleistocene, these islands were often connected as global sea levels dropped following glaciation. Given a continental shelf only 30-60 m below sea level, the terrestrial biota of the Seychelles Archipelago likely dispersed amongst now isolated islands during the Pleistocene. Hypogeophis rostratus is an egg-laying, direct-developing caecilian amphibian found on 10 islands in the granitic Seychelles. Despite the seemingly limited dispersal abilities of this salt-intolerant amphibian, its distribution on multiple islands suggests likely historic dispersal across now submerged continental shelf corridors. We tested for the genetic signature of these historic corridors using fine-scale genomic data (ddRADseq). We found that genomic clusters often did not correspond to islands in the archipelago and that isolation-by-distance patterns were more consistent with gene flow across a continuous landscape than with isolated island populations. Using effective migration surfaces and ancestral range expansion prediction, we found support for contemporary populations originating near the large southern island of Mahé and dispersing to northern islands via the isolated Frégate island, with additional historic migration across the flat expanse of the Seychelles bank. Collectively, our results suggest that biogeographic patterns can retain signals from Pleistocene 'palaeo-islands' and that present-day islands can be thought of as hosting bottlenecks or transient refugia rather than discrete genetic units. Thus, the signatures of gene flow associated with palaeo-islands may be stronger than the isolating effects of contemporary islands in terrestrial species distributed on continental shelf islands.

Biodiversity conservation requires effective monitoring of ecological communities in remote locations, where limited accessibility often restricts survey efforts. Passive acoustic monitoring (PAM) is becoming an established method for measuring biodiversity, facilitated by the increased accessibility of autonomous recording units. Comparing the performance of PAM and traditional survey methods can provide insights into how species characteristics such as foraging strata, vocal behaviour and taxonomy influence the detection ability of each method. Here, avian species occurrences were collected using PAM and point count surveys (PCS) across three forest fragments in the Taita Hills, an Afromontane sky-island in southeastern Kenya. These montane forests contain high species-richness and endemism, but they have been severely fragmented and degraded as a result of deforestation, making comprehensive monitoring critical for conservation. We grouped detected species into clusters based on their foraging strata, using partitioning around medoid cluster analysis. From 25 survey sites, PAM and PCS detected 60 and 57 species, respectively, indicating that PAM is as effective as PCS for surveying montane tropical birds. However, species that primarily foraged on the ground and secondarily used the understorey, and species that primarily used the understorey and secondarily used the canopy, were more likely to be recorded by PAM than by PCS. Species that only used the understorey were more likely to be recorded by PCS. Investigation of broad taxonomic groupings showed that PAM was 20% more likely to record non-passerines than PCS, while passerines were equally likely to be recorded by either method. This study highlights how species characteristics, such as foraging strata and taxonomic group, influence the performance of surveying methods. By identifying which species are best monitored by each method, this study can inform more targeted monitoring strategies, improving accuracy and supporting biodiversity management efforts to mitigate ongoing species loss.

Freshwater ecosystems are globally threatened by habitat loss, pollution, and invasive species, all of which are particularly acute in urban areas. To assess the impacts of urbanization on freshwater biodiversity-specifically the effects of alien species on native primary aquatic vertebrates-we investigated the World Heritage Site, Lake Xochimilco in Mexico City. Focusing on fishes and amphibians, we applied environmental DNA metabarcoding using primer pairs targeting mitochondrial 12S and 16S across the remnant lake and collected 14 aquatic environmental variables for sampled sites. Our survey recovered ca. 60% of Lake Xochimilco's historically recorded fish and amphibian species, including rare species and novel taxa not detected by past traditional surveys. However, our findings imply a severely degraded wetland, with alpha diversity indices indicating a low-diversity ecosystem dominated by alien fishes. Beta diversity analysis revealed a heterogeneous ecosystem that may be driven partially by the presence of alien fish, particularly cyprinids. Environmental variables linked to pollution predicted the presence of non-native fish families. We also found evidence that some species prefer to occupy different water bodies within the lake remnant. Despite the ongoing degradation of this ecosystem, native and endemic fauna are persisting, although detections were typically rare. We found no evidence of the Critically Endangered axolotl salamanders (Ambystoma sp.) from wild sites; however, we detected their presence in one wildlife refuge, highlighting the potential of refuges to prevent complete extinction in the wild. We also found evidence of cryptic taxonomic diversity in Lithobates frogs and evidence of endemic genera, including the threatened mexclapique fish (Girardinicthys viviparus). These fishes are considered extirpated, suggesting remnant populations persist undetected by traditional surveys. Despite clear evidence of an ecosystem under extreme decline compared to historical biological records, our study demonstrates the potential for restoration, given the presence of native freshwater species and the success of wildlife refuges.

The cichlid fishes of East Africa are renowned for their rapid and species-rich adaptive radiations. However, some specialist cichlid lineages in the region have not undergone extensive diversification; it is plausible that these lineages contain cryptic allopatric diversity. The tilapiine cichlid Oreochromis amphimelas (Hilgendorf, 1905) is a distinctive species specialised for high salinity, high alkalinity and high temperature soda lakes in the East African Rift Valley. Here, we investigated variation among O. amphimelas populations using a combination of reduced-representation genome sequences, mitochondrial DNA sequences and morphological data. Genetic data revealed two highly divergent genomic lineages, with no evidence of ongoing gene flow. Specifically, the Lake Manyara population was strongly differentiated from the four other populations studied, which show relatively low levels of genetic differentiation among them. Genetic differentiation between Lake Manyara and the other populations is seen across the genome, as characterised by elevated windowed F ST. Despite the clear genomic divergence between the Lake Manyara and other soda lake populations, there were no apparent morphological differences between the two lineages, indicating they may be considered two cryptic species. It is possible that O. amphimelas lineages have diverged vicariantly following regional geomorphological change. Identification of two potential geographically separated cryptic species in the lineage has conservation implications, given that O. amphimelas is currently categorised as Endangered on the IUCN Red List of Threatened Species, due to threats from fisheries and environmental change.

Aim: Biotic interchanges between Africa, India, and Eurasia are central to explaining the present-day distribution and diversity of freshwater organisms across these landmasses. Synbranchiformes is a diverse and species-rich clade of freshwater acanthomorph fishes found on all southern continents except Antarctica, and include eel- and perch-like, air-breathing and non-air-breathing fishes. Lacking a comprehensive and resolved phylogeny of the entire clade, contemporary interpretations of synbranchiform biogeography invoke scenarios as disparate as Gondwanan vicariance and pan-global rafting to explain their modern-day distribution. Here, we study their biogeographic history of continental dispersal events and test whether these are associated with increases in lineage diversification.Location: Asia, India, Africa freshwater habitats.TaxonSynbranchiformes (gouramis, snakeheads, swamp eels, and relatives).Methods: We used nearly 1000 ultra-conserved elements (UCEs) and Sanger-sequenced genes to infer a phylogeny with representatives of all major synbranchiform lineages and nearly two-thirds of its known species diversity. Incorporating fossil calibrations, we inferred a time-calibrated phylogeny to which we apply Bayesian methods of ancestral area reconstruction and test for diversification rate shifts.Results: Analyses of UCE data provide a resolved phylogeny for major synbranchiform lineages. Divergence times support a most recent common ancestor of the entire clade approximately 79.2 million years ago. We infer significant increases in lineage diversification in both the spiny eels (Mastacembelidae) and the genus Betta (Osphronemidae).Main Conclusions: Our results reject the hypothesis of Gondwanan vicariance explaining synbranchiform biogeography. Instead, our analyses reconstruct a southeast Asian origin of the entire clade and independent dispersal events to other continents by snakeheads, anabantids, and spiny eels, with no signal of elevated lineage diversification occurring after these invasions. Higher lineage diversification rates in spiny eels pre-date their arrival to Africa, while the high diversification rates observed in Betta were initiated prior to the flooding of insular Sundaland in southeast Asia.

ABSTRACTInfectious diseases spread through international wildlife trade networks, presenting major conservation and welfare challenges. The diseases amphibian chytridiomycosis (caused predominantly by chytrid fungus Batrachochytrium dendrobatidis, Bd) and ranavirosis (caused by iridoviruses in the genus Ranavirus, Rv) are the result of infection by globally distributed pathogens. These pathogens spread internationally through live‐animal trade networks and have driven population declines, mass mortalities, and community collapse for a broad range of amphibian species. Environmental (e)DNA methods may provide highly sensitive and non‐invasive pathogen surveillance for traded or wild amphibians. To investigate the relationship between eDNA detection and environmental pathogen persistence, eDNA degradation rates were quantified across a range of temperatures (15°C–25°C) for both Bd and Ranavirus. Estimated decay rates suggest that overall pathogen eDNA concentration degrades by 99% between 18.9–52.4 h. Low levels of pathogen eDNA remained detectable for the duration of the experiment (> 28 days). Time was found to have a significant negative effect on eDNA concentration for both pathogens (p < 0.001). The negative effect of temperature on eDNA concentration was significant for both pathogens (20°C for Rv, p < 0.05; 25°C for Bd/Rv p < 0.001). We argue that high concentrations of eDNA represent viable pathogen in the environment, demonstrating the usefulness of eDNA for the monitoring of disease status of consignments of traded amphibians.

Abstract Adaptive radiation is characterized by eco-morphological differentiation, in which niche partitioning has been shown to be a central response to natural selection during the diversification of animal clades. This process is suggested to have generated the exceptional biodiversity in the East African rift lakes; however, aside from the cichlid fishes, the nature of the divergence, over time, or among species is less clear in the other radiations. To address this, we focus on two distantly related sympatric Lake Tanganyika catfish clades, the genus Synodontis, considered to be Müllerian mimics, and the subfamily Claroteinae. We investigate to what extent, if any, these radiations have undergone eco-morphological diversification. We place these radiations in a common phylogenetic context, and test for morphological divergence and trophic niche partitioning using novel trait data and stable isotope signatures. Diversification of both catfish clades was recent, with the Synodontis radiation synchronized in time with individual genera within the claroteine radiation, suggesting initial diversification was facilitated by lake basin dynamics and/or lake level fluctuations. There is evidence for eco-morphological diversification within the claroteine radiation, as inferred from observed morphological disparity and divergence in diet both between and within genera; however, several species show significant overlap in dietary isotopic signatures. In contrast, the Synodontis radiation has greater overlap between taxa both in morphology and in isotopic signatures potentially indicating niche conservatism, suggesting different selective forces may be driving these radiations.

Unusually for oceanic islands, the granitic Seychelles host multiple lineages of endemic amphibians. This includes an ancient (likely ca. 60 million years) radiation of eight caecilian species, most of which occur on multiple islands. These caecilians have a complicated taxonomic history and their phylogenetic inter-species relationships have been difficult to resolve. Double-digest RAD sequencing (ddRADseq) has been applied extensively to phylogeography and increasingly to phylogenetics but its utility for resolving ancient divergences is less well established. To address this, we applied ddRADseq to generate a genome-wide SNP panel for phylogenomic analyses of the Seychelles caecilians, whose phylogeny has so far not been satisfactorily resolved with traditional DNA markers. Based on 129,154 SNPs, we resolved deep and shallow splits, with strong support. Our findings demonstrate the capability of genome-wide SNPs for evolutionary inference at multiple taxonomic levels and support the recently proposed synonymy of Grandisonia Taylor, 1968 with Hypogeophis Peters, 1879. We revealed three clades of Hypogeophis (large-, medium- and short-bodied) and identify a single origin of the diminutive, stocky-bodied and pointy-snouted phenotype.

Characterizing reproductive barriers such as mating preferences within rapid evolutionary radiations is crucial for understanding the early stages of speciation. Cichlid fishes are well-known for their adaptive radiations and capacity for rapid speciation and as such we investigate assortative mating among Alcolapia species; a recent (<10,000 years), small adaptive radiation, endemic to the extreme soda lakes, Magadi (one species) and Natron (three species), in East Africa. In seminatural aquarium conditions, we observed both courtship and mate choice (tested by microsatellite paternity analysis) to be significantly assortative among the three sympatric Natron species in a three-way choice experiment. This was also the case between allopatric species from Natron and Magadi, as found in a two-way choice experiment. However, the proportion of disassortative matings was substantial in both of these experiments, with hybrids comprising 29% of offspring in sympatric species and 11.4% in allopatric species comparisons. Previous work suggests that the Natron/Magadi split might not be much older than the radiation within Natron, so the similar rate of hybridization in the allopatric comparison is surprising and inconsistent with predictions of reinforcement theory, which predicts a faster rate of accumulation of premating isolation in sympatry. The relatively weak assortative mating in sympatry suggests that additional reproductive barriers, such as microhabitat preferences or spatial structuring may contribute to genetic isolation in nature.

Endeavours in species discovery, particularly the characterisation of cryptic species, have been greatly aided by the application of DNA molecular sequence data to phylogenetic reconstruction and inference of evolutionary and biogeographic processes. However, the extent of cryptic and undescribed diversity remains unclear in tropical freshwaters, where biodiversity is declining at alarming rates. To investigate how data on previously undiscovered biodiversity impacts inferences of biogeography and diversification dynamics, we generated a densely sampled species-level family tree of Afrotropical Mochokidae catfishes (220 valid species) that was ca. 70 % complete. This was achieved through extensive continental sampling specifically targeting the genus Chiloglanis a specialist of the relatively unexplored fast-flowing lotic habitat. Applying multiple species-delimitation methods, we report exceptional levels of species discovery for a vertebrate genus, conservatively delimiting a staggering ca. 50 putative new Chiloglanis species, resulting in a near 80 % increase in species richness for the genus. Biogeographic reconstructions of the family identified the Congo Basin as a critical region in the generation of mochokid diversity, and further revealed complex scenarios for the build-up of continental assemblages of the two most species rich mochokid genera, Synodontis and Chiloglanis. While Syndontis showed most divergence events within freshwater ecoregions consistent with largely in situ diversification, Chiloglanis showed much less aggregation of freshwater ecoregions, suggesting dispersal as a key diversification process in this older group. Despite the significant increase in mochokid diversity identified here, diversification rates were best supported by a constant rate model consistent with patterns in many other tropical continental radiations. While our findings highlight fast-flowing lotic freshwaters as potential hotspots for undescribed and cryptic species diversity, a third of all freshwater fishes are currently threatened with extinction, signifying an urgent need to increase exploration of tropical freshwaters to better characterise and conserve its biodiversity.

Extant neosuchian crocodiles are represented by only 24 taxa that are confined to the tropics and subtropics. However, at other intervals during their 200 Myr evolutionary history the clade reached considerably higher levels of species-richness, matched by more widespread distributions. Neosuchians have occupied numerous habitats and niches, ranging from dwarf riverine forms to large marine predators. Despite numerous previous studies, several unsolved questions remain with respect to their biogeographic history, including the geographical origins of major groups, e.g. Eusuchia and Neosuchia itself. We carried out the most comprehensive biogeographic analysis of Neosuchia to date, based on a multivariate K-means clustering approach followed by the application of two ancestral area estimation methods (BioGeoBEARS and Bayesian ancestral location estimation) applied to two recently published phylogenies. Our results place the origin of Neosuchia in northwestern Pangaea, with subsequent radiations into Gondwana. Eusuchia probably emerged in the European archipelago during the Late Jurassic/Early Cretaceous, followed by dispersals to the North American and Asian landmasses. We show that putative transoceanic dispersal events are statistically significantly less likely to happen in alligatoroids. This finding is consistent with the saltwater intolerant physiology of extant alligatoroids, bolstering inferences of such intolerance in their ancestral lineages.

Uropeltidae is a clade of small fossorial snakes (ca. 64 extant species) endemic to peninsular India and Sri Lanka. Uropeltid taxonomy has been confusing, and the status of some species has not been revised for over a century. Attempts to revise uropeltid systematics and undertake evolutionary studies have been hampered by incompletely sampled and incompletely resolved phylogenies. To address this issue, we take advantage of historical museum collections, including type specimens, and apply genome-wide shotgun (GWS) sequencing, along with recent field sampling (using Sanger sequencing) to establish a near-complete multilocus species-level phylogeny (ca. 87% complete at species level). This results in a phylogeny that supports the monophyly of all genera (if Brachyophidium is considered a junior synonym of Teretrurus), and provides a firm platform for future taxonomic revision. Sri Lankan uropeltids are probably monophyletic, indicating a single colonisation event of this island from Indian ancestors. However, the position of Rhinophis goweri (endemic to Eastern Ghats, southern India) is unclear and warrants further investigation, and evidence that it may nest within the Sri Lankan radiation indicates a possible recolonisation event. DNA sequence data and morphology suggest that currently recognised uropeltid species diversity is substantially underestimated. Our study highlights the benefits of integrating museum collections in molecular genetic analyses and their role in understanding the systematics and evolutionary history of understudied organismal groups.

Long-term monitoring is critical to measure the response of biodiversity patterns and processes to human-mediated environmental pressures. This is particularly pertinent in freshwaters, where recent estimates indicated a third of all fish species are threatened with extinction, making ongoing biomonitoring essential for conservation management. High frequency annual monitoring is critical for identifying temporal changes in fish community composition; however, traditional survey methods are typically less practical over such timeframes. While environmental (e)DNA measurement represents a potentially powerful tool for monitoring temporal community dynamics, studies are lacking. To address this deficit, we generated a high frequency time-series dataset of entire fish communities using eDNA metabarcoding, to directly assess the repeatability and sensitivity of this method for detecting annual population trends. We targeted two differing environments (freshwater vs. intertidal) within the Thames catchment, UK, where detailed historical records from traditional monitoring were available for comparison. To test how robust eDNA data is for inferring the known community, we applied a hierarchical, nested design encompassing short and longer-term variation in eDNA data. Our analyses showed that irrespective of environment, eDNA metabarcoding represented known seasonal shifts in fish communities, where increased relative read abundance of eDNA coincided with known migratory and spawning events, including those of the critically endangered native species Anguilla anguilla (European eel). eDNA species detections across a single year included over 75% of species recorded in a ca. 30-year historical dataset, highlighting the power of eDNA for species detection. Our findings provide greater insight into the utility of eDNA metabarcoding for recovering temporal trends in fish communities from dynamic freshwater systems and insight into the potential best sampling strategy for future eDNA surveys.

Clade ages within the crocodylomorph clade Neosuchia have long been debated. Molecular and morphological studies have yielded remarkably divergent results. Despite recent advances, there has been no comprehensive relative comparison of the major time calibration methods available to estimate clade ages based on morphological data. We used four methods (cal3, extended Hedman, smoothed ghost lineage analysis (sGLA) and the fossilized birth–death model (FBD)) to date clade ages derived from a published crocodylomorph supertree and a new neosuchian phylogeny. All time‐scaling methods applied here agree on the origination of Neosuchia during the Late Triassic or Early Jurassic, and the presence of the major extant eusuchian groups (Crocodyloidea, Gavialoidea, Alligatoroidea and Caimaininae) by the end of the Late Cretaceous. The number of distinct lineages present before the K/Pg boundary is less certain, with support for two competing scenarios in which Crocodylinae, Tomistominae and Diplocynodontinae either: (1) diverged from other eusuchian lineages before the K/Pg boundary; or (2) evolved during a ‘burst’ of diversification after the K/Pg event. Cal3 and FBD proved to be the most suitable methods for time‐scaling phylogenetic trees dominated by fossil taxa. Extended Hedman estimates are substantially older than the others, with larger standard deviations and a strong sensitivity to taxon sampling and topological changes; sGLA has similar problems. We conclude that a detailed understanding of phylogenetic relationships, tree reconstruction methods, and good taxonomic coverage (in particular the inclusion of the oldest taxon in each clade) is essential when evaluating the results of such dating analyses.

AbstractWhile many studies have considered the ability of eDNA to assess animal communities in lacustrine settings, fewer have considered riverine systems, particularly those spanning the environmental gradients present in large river basins. Such dynamic systems are challenging for eDNA biomonitoring due to differing eDNA transport distances in rivers and the effects of river chemistry. To address this challenge, we focused on the Thames River system, UK, which has exceptional historical fish records providing a baseline to test the accuracy of eDNA metabarcoding in recovering fish community structure across both fresh and tidal zones. Two primer sets targeting 12S and CO1 regions were used to capture fish communities across the Thames catchment, from the upper freshwaters to the mid estuary. eDNA was collected at 35 sites, 14 of which were simultaneously paired with traditional fish surveys for direct comparison. We demonstrated that eDNA metabarcoding consistently detected more freshwater species than traditional methods, despite extensive sampling effort using the latter. In contrast, metabarcoding did not perform as well as traditional approaches in estuarine waters, although results included the novel detection of the protected sea lamprey. We further demonstrated that minor variations in the recovery of all approaches would not impact on the assessment of simple ecological models of community structure and, thus, some variability between approaches should not be viewed as a serious hindrance to uptake. Rather, our findings support a growing consensus that eDNA can reliably detect fish communities across dynamic freshwater habitats.

Phylogenetic relationships of sub-Saharan African natricine snakes are understudied and poorly understood, which in turn has precluded analyses of the historical biogeography of the Seychelles endemic Lycognathophis seychellensis. We inferred the phylogenetic relationships of Seychelles and mainland sub-Saharan natricines by analysing a multilocus DNA sequence dataset for three mitochondrial (mt) and four nuclear (nu) genes. The mainland sub-Saharan natricines and L. seychellensis comprise a well-supported clade. Two maximally supported sets of relationships within this clade are (Limnophis,Natriciteres) and (Afronatrix,(Hydraethiops,Helophis)). The relationships of L. seychellensis with respect to these two lineages are not clearly resolved by analysing concatenated mt and nu data. Analysed separately, nu data best support a sister relationship of L. seychellensis with (Afronatrix,(Hydraethiops,Helophis)) and mt data best support a sister relationship with all mainland sub-Saharan natricines. Methods designed to cope with incomplete lineage sorting strongly favour the former hypothesis. Genetic variation among up to 33 L. seychellensis from five Seychelles islands is low. Fossil calibrated divergence time estimates support an overseas dispersal of the L. seychellensis lineage to the Seychelles from mainland Africa ca. 43-25 million years before present (Ma), rather than this taxon being a Gondwanan relic.

AbstractBackground and AimsEnvironmental DNA (eDNA) metabarcoding provides a highly sensitive method of surveying freshwater fish communities, although studies to date have largely been restricted to temperate ecosystems. Due to limited reference sequence availability and challenges identifying closely related and rare species in diverse tropical ecosystems, the effectiveness of metabarcoding methods for surveying tropical fish communities from eDNA samples remains uncertain. To address this, we applied an eDNA metabarcoding approach to survey Lake Tanganyika's (LT) species‐rich littoral fish communities.Materials and MethodsAs this system contains many closely related species, particularly cichlid fishes, we used four primer sets including a cichlid‐specific primer set (Cichlid_CR). A reference database was built for the 12s, 16s, and control region for 358 fish species including over 93% of known cichlids.Results and DiscussionIn silico and in situ results demonstrated wide variability in the taxonomic resolution of assignments by each primer with the cichlid‐specific marker (Cichlid_CR) enabling greater species‐level assignments for this highly diverse family. A greater number of non‐cichlid teleost species were detected at sites compared to the visual survey data. For cichlid species however, sequencing depth substantially influenced species richness estimates obtained from eDNA samples, with increased depths producing estimates comparable to that obtained from the visual survey data. ConclusionsOur study highlights the importance of sequencing depth and local reference databases when undertaking metabarcoding studies within diverse ecosystems, as well as demonstrating the potential of eDNA metabarcoding for surveying diverse tropical fish communities, even those containing closely related species within evolutionary radiations.