Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome.

In arthropod-associated microbial communities, insect-specific viruses (ISVs) are prevalent yet understudied due to limited infectivity outside their natural hosts. However, ISVs might play a crucial role in regulating mosquito populations and influencing arthropod-borne virus transmission. Some studies have indicated a core virome in mosquitoes consisting of mostly ISVs. Employing single mosquito metagenomics, we comprehensively profiled the virome of native and invasive mosquito species in Belgium. This approach allowed for accurate host species determination, prevalence assessment of viruses and Wolbachia, and the identification of novel viruses. Contrary to our expectations, no abundant core virome was observed in Culex mosquitoes from Belgium. In that regard, we caution against rigidly defining mosquito core viromes and encourage nuanced interpretations of other studies. Nonetheless, our study identified 45 viruses of which 28 were novel, enriching our understanding of the mosquito virome and ISVs. We showed that the mosquito virome in this study is species-specific and less dependent on the location where mosquitoes from the same species reside. In addition, because Wolbachia has previously been observed to influence arbovirus transmission, we report the prevalence of Wolbachia in Belgian mosquitoes and the detection of several Wolbachia mobile genetic elements. The observed prevalence ranged from 83% to 92% in members from the Culex pipiens complex.IMPORTANCECulex pipiens mosquitoes are important vectors for arboviruses like West Nile virus and Usutu virus. Virome studies on individual Culex pipiens, and on individual mosquitoes in general, have been lacking. To mitigate this, we sequenced the virome of 190 individual Culex and 8 individual Aedes japonicus mosquitoes. We report the lack of a core virome in these mosquitoes from Belgium and caution the interpretation of other studies in this light. The discovery of new viruses in this study will aid our comprehension of insect-specific viruses and the mosquito virome in general in relation to mosquito physiology and mosquito population dynamics.

Expanding the swimmer's itch pool of the Benelux: a first record of the neurotropic Trichobilharzia regenti and potential link to human infection.

BACKGROUND: Swimmer's itch, an allergic contact dermatitis caused by avian and mammalian blood flukes, is a parasitic infection affecting people worldwide. In particular, avian blood flukes of the genus Trichobilharzia are infamous for their role in swimmer's itch cases. These parasites infect waterfowl as a final host, but incidental infections by cercariae in humans are frequently reported. Upon accidental infections of humans, parasite larvae will be recognized by the immune system and destroyed, leading to painful itchy skin lesions. However, one species, Trichobilharzia regenti, can escape this response in experimental animals and reach the spinal cord, causing neuroinflammation. In the last few decades, there has been an increase in case reports across Europe, making it an emerging zoonosis. METHODS: Following a reported case of swimmer's itch in Kampenhout in 2022 (Belgium), the transmission site consisting of a private pond and an adjacent creek was investigated through a malacological and parasitological survey. RESULTS: Six snail species were collected, including the widespread Ampullaceana balthica, a well-known intermediate host for Trichobilharzia parasites. Shedding experiments followed by DNA barcoding revealed a single snail specimen to be infected with T. regenti, a new species record for Belgium and by extension the Benelux. Moreover, it is the most compelling case to date of the link between this neurotropic parasite and cercarial dermatitis. Additionally, an Echinostomatidae sp. and Notocotylus sp. were isolated from two other specimens of A. balthica. However, the lack of reference DNA sequences for these groups in the online repositories prevented genus- and species-level identification, respectively. CONCLUSIONS: The presence of T. regenti in Belgium might have severe clinical implications and its finding highlights the need for increased vigilance and diagnostic awareness among medical professionals. The lack of species-level identification of the other two parasite species showcases the barcoding void for trematodes. Overall, these findings demonstrate the need for a Belgian framework to rapidly detect and monitor zoonotic outbreaks of trematode parasites within the One Health context.

Publishing data to support the fight against human vector-borne diseases.

Vector-borne diseases are responsible for more than 17% of human cases of infectious diseases. In most situations, effective control of debilitating and deadly vector-bone diseases (VBDs), such as malaria, dengue, chikungunya, yellow fever, Zika and Chagas requires up-to-date, robust and comprehensive information on the presence, diversity, ecology, bionomics and geographic spread of the organisms that carry and transmit the infectious agents. Huge gaps exist in the information related to these vectors, creating an essential need for campaigns to mobilise and share data. The publication of data papers is an effective tool for overcoming this challenge. These peer-reviewed articles provide scholarly credit for researchers whose vital work of assembling and publishing well-described, properly-formatted datasets often fails to receive appropriate recognition. To address this, GigaScience's sister journal GigaByte partnered with the Global Biodiversity Information Facility (GBIF) to publish a series of data papers, with support from the Special Programme for Research and Training in Tropical Diseases (TDR), hosted by the World Health Organisation (WHO). Here we outline the initial results of this targeted approach to sharing data and describe its importance for controlling VBDs and improving public health.

DNA barcoding echinoderms from the East Coast of South Africa. The challenge to maintain DNA data connected with taxonomy.

Echinoderms are marine water invertebrates that are represented by more than 7000 extant species, grouped in five classes and showing diverse morphologies (starfish, sea lilies, feather stars, sea urchins, sea cucumbers, brittle and basket stars). In an effort to further study their diversity, DNA barcodes (DNA fragments of the 5' end of the cytochrome c oxidase subunit I gene, COI) have been used to complement morphological examination in identifying evolutionary lineages. Although divergent clusters of COI sequences were reported to generally match morphological species delineations, they also revealed some discrepancies, suggesting overlooked species, ecophenotypic variation or multiple COI lineages within one species. Here, we sequenced COI fragments of 312 shallow-water echinoderms of the East Coast of South Africa (KwaZulu-Natal Province) and compared morphological identifications with species delimitations obtained with four methods that are exclusively based on COI sequences. We identified a total of 103 morphospecies including 18 that did not exactly match described species. We also report 46 COI sequences that showed large divergences (>5% p-distances) with those available to date and publish the first COI sequences for 30 species. Our analyses also identified discordances between morphological identifications and COI-based species delimitations for a considerable proportion of the morphospecies studied here (49/103). For most of them, further investigation is necessary to keep a sound connection between taxonomy and the growing importance of DNA-based research.

Large-scale and small-scale population genetic structure of the medically important gastropod species Bulinus truncatus (Gastropoda, Heterobranchia).

BACKGROUND: Gastropod snails remain strongly understudied, despite their important role in transmitting parasitic diseases. Knowledge of their distribution and population dynamics increases our understanding of the processes driving disease transmission. We report the first study to use high-throughput sequencing (HTS) to elucidate the population genetic structure of the hermaphroditic snail Bulinus truncatus (Gastropoda, Heterobranchia) on a regional (17-150 km) and inter-regional (1000-5400 km) scale. This snail species acts as an intermediate host of Schistosoma haematobium and Schistosoma bovis, which cause human and animal schistosomiasis respectively. METHODS: Bulinus truncatus snails were collected in Senegal, Cameroon, Egypt and France and identified through DNA barcoding. A single-end genotyping-by-sequencing (GBS) library, comprising 87 snail specimens from the respective countries, was built and sequenced on an Illumina HiSeq 2000 platform. Reads were mapped against S. bovis and S. haematobium reference genomes to identify schistosome infections, and single nucleotide polymorphisms (SNPs) were scored using the Stacks pipeline. These SNPs were used to estimate genetic diversity, assess population structure and construct phylogenetic trees of B. truncatus. RESULTS: A total of 10,750 SNPs were scored and used in downstream analyses. The phylogenetic analysis identified five clades, each consisting of snails from a single country but with two distinct clades within Senegal. Genetic diversity was low in all populations, reflecting high selfing rates, but varied between locations due to habitat variability. Significant genetic differentiation and isolation by distance patterns were observed at both spatial scales, indicating that gene flow is not strong enough to counteract the effects of population bottlenecks, high selfing rates and genetic drift. Remarkably, the population genetic differentiation on a regional scale (i.e. within Senegal) was as large as that between populations on an inter-regional scale. The blind GBS technique was able to pick up parasite DNA in snail tissue, demonstrating the potential of HTS techniques to further elucidate the role of snail species in parasite transmission. CONCLUSIONS: HTS techniques offer a valuable toolbox to further investigate the population genetic patterns of intermediate schistosome host snails and the role of snail species in parasite transmission.

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing.

BACKGROUND: The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species' biology, behavior, and dispersal patterns were poorly investigated to date. METHODOLOGY/PRINCIPAL FINDINGS: To understand the species' population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. CONCLUSIONS/SIGNIFICANCE: Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of 'Single Nucleotide Polymorphism' markers. Considering its public health importance, it is crucial to further investigate the species' population genetic dynamic, including a larger sampling and additional genomic markers.

From a long-distance threat to the invasion front: a review of the invasive Aedes mosquito species in Belgium between 2007 and 2020.

Invasive mosquito species (IMS) and their associated mosquito-borne diseases are emerging in Europe. In Belgium, the first detection of Aedes albopictus (Skuse 1894) occurred in 2000 and of Aedes japonicus japonicus (Theobald 1901) in 2002. Early detection and control of these IMS at points of entry (PoEs) are of paramount importance to slow down any possible establishment. This article reviews the introductions and establishments recorded of three IMS in Belgium based on published (2007-2014) and unpublished (2015-2020) data collected during several surveillance projects. In total, 52 PoEs were monitored at least once for the presence of IMS between 2007 and 2020. These included used tyre and lucky bamboo import companies, airports, ports, parking lots along highways, shelters for imported cutting plants, wholesale markets, industrial areas, recycling areas, cemeteries and an allotment garden at the country border with colonised areas. In general, monitoring was performed between April and November. Mosquitoes were captured with adult and oviposition traps as well as by larval sampling. Aedes albopictus was detected at ten PoEs, Ae. japonicus at three PoEs and Aedes koreicus (Edwards 1917) at two PoEs. The latter two species have established overwintering populations. The percentage of PoEs positive for Ae. albopictus increased significantly over years. Aedes albopictus is currently entering Belgium through lucky bamboo and used tyre trade and passive ground transport, while Ae. japonicus through used tyre trade and probably passive ground transport. In Belgium, the import through passive ground transport was first recorded in 2018 and its importance seems to be growing. Belgium is currently at the invasion front of Ae. albopictus and Ae. japonicus. The surveillance and control management actions at well-known PoEs associated to long-distance introductions are more straightforward than at less-defined PoEs associated with short-distance introductions from colonised areas. These latter PoEs represent a new challenge for IMS management in Belgium in the coming years. Aedes albopictus is expected to become established in Belgium in the coming years, hence increasing the likelihood of local arbovirus transmission. The implementation of a sustainable, structured and long-term IMS management programme, integrating active and passive entomological surveillance, vector control and Public Health surveillance is therefore pivotal.

Two fatal autochthonous cases of airport malaria, Belgium, 2020.

We report an outbreak investigation of two fatal cases of autochthonous Plasmodium falciparum malaria that occurred in Belgium in September 2020. Various hypotheses of the potential source of infection were investigated. The most likely route of transmission was through an infectious exotic Anopheles mosquito that was imported via the international airport of Brussels or the military airport Melsbroek and infected the cases who lived at 5 km from the airports. Based on genomic analysis of the parasites collected from the two cases, the most likely origin of the Plasmodium was Gabon or Cameroon. Further, the parasites collected from the two Belgian patients were identical by descent, which supports the assumption that the two infections originated from the bite of the same mosquito, during interrupted feeding. Although airport malaria remains a rare event, it has significant implications, particularly for the patient, as delayed or missed diagnosis of the cause of illness often results in complications and mortality. Therefore, to prevent such severe or fatal outcomes, we suggest a number of public health actions including increased awareness among health practitioners, especially those working in the vicinity of airports, and increased surveillance of exotic mosquito species at airports.

MEMO: Monitoring of exotic mosquitoes in Belgium.

'MEMO: Monitoring of Exotic MOsquitoes in Belgium' is a sampling event dataset published by the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. It forms part of the early detection of exotic mosquito species (EMS) along high-risk introduction routes in Belgium, where data are collected at defined points of entry (PoEs) using a standardised protocol. The MEMO dataset contains mosquito sampling counts performed between 2017 and 2020. MEMO+2020, an extension of the MEMO dataset, contains only Aedes albopictus mosquito trap counts performed in 2020. Here, we present these data published as a standardised Darwin Core archive, which includes, for each sampling event, an eventID, date, location and sampling protocol (in the event core); and an occurrenceID for each occurrence (tube), the number of collected individuals per tube, species status (present/absent), information on the identification and scientific name (in the occurrence extension).

First Detections of Culiseta longiareolata (Diptera: Culicidae) in Belgium and the Netherlands.

Culiseta (Allotheobaldia) longiareolata (Macquart) (Diptera: Culicidae) is an ornithophilic mosquito species that occurs in the southern Palaearctic Region from the Azores to Central Asia, the Ethiopian Region, India, and Pakistan. Although it has a widespread distribution range, the species was only recently reported in Western and Central Europe. Between 2017 and 2020, larvae, pupae, and adults of Cs. longiareolata (n = 161) were found at 13 distinct locations in Belgium (n = 4) and The Netherlands (n = 9). Collected mosquitoes were morphologically identified and the identification was then validated by COI DNA barcoding. These are the first records of the species in the above-mentioned countries. The present results suggest that Cs. longiareolata could be increasing its distribution range in temperate regions, indicating a warming climate. As the species might be a potential vector of bird pathogens (e.g., West Nile virus), its spread in Western Europe is noteworthy.

Population genetic structure of the Asian bush mosquito, Aedes japonicus (Diptera, Culicidae), in Belgium suggests multiple introductions.

BACKGROUND: Aedes japonicus japonicus has expanded beyond its native range and has established in multiple European countries, including Belgium. In addition to the population located at Natoye, Belgium, locally established since 2002, specimens were recently collected along the Belgian border. The first objective of this study was therefore to investigate the origin of these new introductions, which were assumed to be related to the expansion of the nearby population in western Germany. Also, an intensive elimination campaign was undertaken at Natoye between 2012 and 2015, after which the species was declared to be eradicated. This species was re-detected in 2017, and thus the second objective was to investigate if these specimens resulted from a new introduction event and/or from a few undetected specimens that escaped the elimination campaign. METHODS: Population genetic variation at nad4 and seven microsatellite loci was surveyed in 224 and 68 specimens collected in Belgium and Germany, respectively. German samples were included as reference to investigate putative introduction source(s). At Natoye, 52 and 135 specimens were collected before and after the elimination campaign, respectively, to investigate temporal changes in the genetic composition and diversity. RESULTS: At Natoye, the genotypic microsatellite make-up showed a clear difference before and after the elimination campaign. Also, the population after 2017 displayed an increased allelic richness and number of private alleles, indicative of new introduction(s). However, the Natoye population present before the elimination programme is believed to have survived at low density. At the Belgian border, clustering results suggest a relation with the western German population. Whether the introduction(s) occur via passive human-mediated ground transport or, alternatively, by natural spread cannot be determined yet from the dataset. CONCLUSION: Further introductions within Belgium are expected to occur in the near future, especially along the eastern Belgian border, which is at the front of the invasion of Ae. japonicus towards the west. Our results also point to the complexity of controlling invasive species, since 4 years of intense control measures were found to be not completely successful at eliminating this exotic at Natoye.

First record of the West Nile virus bridge vector Culex modestus Ficalbi (Diptera: Culicidae) in Belgium, validated by DNA barcoding.

A thorough knowledge of the presence and spatio-temporal distribution patterns of vector species are pivotal to assess the risk of mosquito-borne diseases in Europe. In 2018, a Culex larva was collected during routine monitoring activities to intercept exotic Aedes mosquito species in the port of Antwerp (Kallo, Belgium). The larva, collected from a pond in mid-September, was morphologically identified as Culex modestus, and this identification was subsequently confirmed by COI barcoding. It is the first confirmed record of this West Nile virus bridge vector in Belgium. The present study also demonstrates the value of DNA-based identification techniques to validate the presence of potential vector species.

High Aedes spp. larval indices in Kinshasa, Democratic Republic of Congo.

BACKGROUND: Dengue, yellow fever, chikungunya and Zika are among the most important emerging infectious vector-borne diseases worldwide. In the Democratic Republic of Congo (DRC), increases in cases of dengue and outbreaks of yellow fever and chikungunya have been reported since 2010. The main vectors of these arboviruses, Aedes aegypti and Aedes albopictus, have been reported in DRC, but there is a lack of detailed information on their presence and spread to guide disease control efforts. METHODS: In 2018, two cross-sectional surveys were conducted in Kinshasa province (DRC), one in the rainy (January/February) and one in the dry season (July). Four hundred houses were visited in each of the four selected communes (N'Djili, Mont Ngafula, Lingwala and Kalamu). Within the peri-domestic area of each household, searches were conducted for larval habitats, which were then surveyed for the presence of Aedes larvae and pupae. A subset of the immature specimens were reared to adults for morphological identification followed by DNA barcoding of the specimens to validate identifications. RESULTS: The most rural commune (Mont Ngafula) had the highest pupal index (number of Aedes spp. pupae per 100 inspected houses) at 246 (20) pupae/100 houses, and Breteau index (BI; number of containers positive for immature stages of Aedes spp. per 100 households) at 82.2 (19.5) positive containers/100 houses for the rainy (and dry) season, respectively. The BI was 21.5 (4.7), 36.7 (9.8) and 41.7 (7.5) in Kalamu, Lingwala and N'Djili in the rainy (and dry) season, respectively. The house index (number of houses positive for at least one container with immature stages of Aedes spp. per 100 inspected houses) was, on average, across all communes, 27.5% (7.6%); and the container index (number of containers positive for immature stages of Aedes spp. per 100 inspected containers) was 15.0% (10.0%) for the rainy (and dry) season, respectively. The vast majority of Aedes-positive containers were found outside the houses [adjusted odds ratio 27.4 (95% confidence interval 14.9-50.1)]. During the dry season, the most productive containers were the ones used for water storage, whereas in the rainy season rubbish and tires constituted key habitats. Both Ae. aegypti and Ae. albopictus were found. Anopheles larvae were found in different types of Aedes larval habitats, especially during the rainy season. CONCLUSIONS: In both surveys and in all communes, the larval indices (BI) were higher than the arbovirus transmission threshold values established by the World Health Organization. Management strategies for controlling Aedes in Kinshasa need to target the key types of containers for Aedes larvae, which are mainly located in outdoor spaces, for larval habitat destruction or reduction.

Detection of Exotic Mosquito Species (Diptera: Culicidae) at International Airports in Europe.

In Europe, the air-borne accidental introduction of exotic mosquito species (EMS) has been demonstrated using mosquito surveillance schemes at Schiphol International Airport (Amsterdam, The Netherlands). Based upon these findings and given the increasing volume of air transport movements per year, the establishment of EMS after introduction via aircraft is being considered a potential risk. Here we present the airport surveillance results performed by the Centre for Monitoring of Vectors of the Netherlands, by the Monitoring of Exotic Mosquitoes (MEMO) project in Belgium, and by the Public Health England project on invasive mosquito surveillance. The findings of our study demonstrate the aircraft mediated transport of EMS into Europe from a wide range of possible areas in the world. Results show accidental introductions of Aedes aegypti and Ae. albopictus, as well as exotic Anopheles and Mansonia specimens. The findings of Ae. albopictus at Schiphol airport are the first evidence of accidental introduction of the species using this pathway in Europe. Furthermore, our results stress the importance of the use of molecular tools to validate the morphology-based species identifications. We recommend monitoring of EMS at airports with special attention to locations with a high movement of cargo and passengers.

A genome-wide data assessment of the African lion (Panthera leo) population genetic structure and diversity in Tanzania.

The African lion (Panthera leo), listed as a vulnerable species on the IUCN Red List of Threatened Species (Appendix II of CITES), is mainly impacted by indiscriminate killing and prey base depletion. Additionally, habitat loss by land degradation and conversion has led to the isolation of some subpopulations, potentially decreasing gene flow and increasing inbreeding depression risks. Genetic drift resulting from weakened connectivity between strongholds can affect the genetic health of the species. In the present study, we investigated the evolutionary history of the species at different spatiotemporal scales. Therefore, the mitochondrial cytochrome b gene (N = 128), 11 microsatellites (N = 103) and 9,103 SNPs (N = 66) were investigated in the present study, including a large sampling from Tanzania, which hosts the largest lion population among all African lion range countries. Our results add support that the species is structured into two lineages at the continental scale (West-Central vs East-Southern), underlining the importance of reviewing the taxonomic status of the African lion. Moreover, SNPs led to the identification of three lion clusters in Tanzania, whose geographical distributions are in the northern, southern and western regions. Furthermore, Tanzanian lion populations were shown to display good levels of genetic diversity with limited signs of inbreeding. However, their population sizes seem to have gradually decreased in recent decades. The highlighted Tanzanian African lion population genetic differentiation appears to have resulted from the combined effects of anthropogenic pressure and environmental/climatic factors, as further discussed.

Genetic structure of fragmented southern populations of African Cape buffalo (Syncerus caffer caffer).

BACKGROUND: African wildlife experienced a reduction in population size and geographical distribution over the last millennium, particularly since the 19th century as a result of human demographic expansion, wildlife overexploitation, habitat degradation and cattle-borne diseases. In many areas, ungulate populations are now largely confined within a network of loosely connected protected areas. These metapopulations face gene flow restriction and run the risk of genetic diversity erosion. In this context, we assessed the "genetic health" of free ranging southern African Cape buffalo populations (S.c. caffer) and investigated the origins of their current genetic structure. The analyses were based on 264 samples from 6 southern African countries that were genotyped for 14 autosomal and 3 Y-chromosomal microsatellites. RESULTS: The analyses differentiated three significant genetic clusters, hereafter referred to as Northern (N), Central (C) and Southern (S) clusters. The results suggest that splitting of the N and C clusters occurred around 6000 to 8400 years ago. Both N and C clusters displayed high genetic diversity (mean allelic richness (A r ) of 7.217, average genetic diversity over loci of 0.594, mean private alleles (P a ) of 11), low differentiation, and an absence of an inbreeding depression signal (mean F IS = 0.037). The third (S) cluster, a tiny population enclosed within a small isolated protected area, likely originated from a more recent isolation and experienced genetic drift (F IS = 0.062, mean A r = 6.160, P a = 2). This study also highlighted the impact of translocations between clusters on the genetic structure of several African buffalo populations. Lower differentiation estimates were observed between C and N sampling localities that experienced translocation over the last century. CONCLUSIONS: We showed that the current genetic structure of southern African Cape buffalo populations results from both ancient and recent processes. The splitting time of N and C clusters suggests that the current pattern results from human-induced factors and/or from the aridification process that occurred during the Holocene period. The more recent S cluster genetic drift probably results of processes that occurred over the last centuries (habitat fragmentation, diseases). Management practices of African buffalo populations should consider the micro-evolutionary changes highlighted in the present study.

Pan-African genetic structure in the African buffalo (Syncerus caffer): investigating intraspecific divergence.

The African buffalo (Syncerus caffer) exhibits extreme morphological variability, which has led to controversies about the validity and taxonomic status of the various recognized subspecies. The present study aims to clarify these by inferring the pan-African spatial distribution of genetic diversity, using a comprehensive set of mitochondrial D-loop sequences from across the entire range of the species. All analyses converged on the existence of two distinct lineages, corresponding to a group encompassing West and Central African populations and a group encompassing East and Southern African populations. The former is currently assigned to two to three subspecies (S. c. nanus, S. c. brachyceros, S. c. aequinoctialis) and the latter to a separate subspecies (S. c. caffer). Forty-two per cent of the total amount of genetic diversity is explained by the between-lineage component, with one to seventeen female migrants per generation inferred as consistent with the isolation-with-migration model. The two lineages diverged between 145 000 to 449 000 years ago, with strong indications for a population expansion in both lineages, as revealed by coalescent-based analyses, summary statistics and a star-like topology of the haplotype network for the S. c. caffer lineage. A Bayesian analysis identified the most probable historical migration routes, with the Cape buffalo undertaking successive colonization events from Eastern toward Southern Africa. Furthermore, our analyses indicate that, in the West-Central African lineage, the forest ecophenotype may be a derived form of the savanna ecophenotype and not vice versa, as has previously been proposed. The African buffalo most likely expanded and diverged in the late to middle Pleistocene from an ancestral population located around the current-day Central African Republic, adapting morphologically to colonize new habitats, hence developing the variety of ecophenotypes observed today.