Phylogenetic Analyses of Cyprinid Species from the Rokel River Basin of Sierra Leone, West Africa: Taxonomic, Biogeographic, and Conservation Implications
1
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
2
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
3
Center for Plant Science Innovation and Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(4), 299; https://0-doi-org.brum.beds.ac.uk/10.3390/d14040299
Submission received: 23 December 2021
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Revised: 1 April 2022
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Accepted: 11 April 2022
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Published: 15 April 2022
(This article belongs to the Special Issue Molecular Evolution and Conservation of Tropical Biodiversity)
Abstract
:The Rokel River (RR) basin is one of the most neglected ichthyofaunal basins, despite the potential for undetected diversity and high levels of endemism. Data on the molecular phylogeny of freshwater fish from this river are rare. Morphological features alone are inadequate for precise species identification. Here, a phylogenetic analysis performed based on the mtDNA Cytb gene for eleven cyprinid fish from the RR basin recovered eleven distinct lineages. The same was also observed for two of our species delineation analyses, of which four are identical to six morphospecies, one is of taxonomic uncertainty, and the rest are currently unrecognized. The disjunct distribution found here in some cyprinid species from the RR basin and their sister species suggests that this river had a past complex historical inter-basin connection exchange with the nearby river basins of the Zaire and lower Guinean ecoregions. The unrecognized diversity observed from cyprinid species of this area may have significant implications for the conservation of biodiversity.
1. Introduction
The Rokel River (RR) is positioned in the northern portion of Sierra Leone with an elevation between 950 m in the northeastern highlands and less than 7 m in the western coastal lowlands [1,2]. It has its source from the Fouta Djallon Highlands in the upper Guinean ecoregion. This river is a hotspot of fish diversity and endemism [1,3,4], as it harbors a total of about 72 freshwater fish, 17 of which are endemic to this river [1,3]. The high rate of endemism would have been facilitated by vicariant events that allowed geodispersal and successive divergence from ancestral populations [5,6,7,8,9]. The ancient connections of many of these smaller coastal rivers or streams in the Guinean ecoregions remain unclear, however recent studies from neighboring countries are beginning to offer some insight into the evolution of these drainages [9,10,11,12].
Fish species’ identification from the RR basin, like all other freshwater systems in Sierra Leone, is poorly explored. The available checklist of freshwater fish of this river basin and the entire country is based on literature records [1,3]. Furthermore, existing morphological and molecular data were combined to decipher unappreciated diversity, which has contributed to the discovery of new species and the re-identification of formerly described species [10,11,12]. Phylogenetic analyses of freshwater fish of this area and the entire country have not been carried out.
Previously, molecular work from neighboring basins has shown that many currently identified widespread species in the Guinean ecoregions consist of individuals from genetically divergent lineages [9,13,14,15]. It has been shown that the morphological approach alone is insufficient to uncover species of the cyprinid genus Enteromius [13,14]. An integrative taxonomy approach is essential to understand fish species diversity and endemism of poorly sampled rivers of the upper Guinean ecoregion, particularly the RR basin [10,11,12,13,14,15,16].
Considering this knowledge gap, there is a critical need for a better understanding of freshwater fish of the RR basin. Documentation of species diversity is vital for fish conservation in this river and could also help in the sustainable management of freshwater systems in the country. The current conservation strategy plan for the upper Guinean Rain Forest focuses on the terrestrial environment, including birds, plants, amphibians, and chimpanzees, with little or no attention on its aquatic systems, particularly on fish species [17]. Certainly, documentation of unknown diversity such as in this study is a key to achieve conservation goals [17].
The present view of diversification within the freshwater system of the RR basin or upper Guinea ecoregion is mainly centered on faunal records [1,4]. These faunal records were a compounding of diverse collections from checklists of individual drainages in the area [3,4] and the Checklist of the Freshwater Fish of Africa [3]. Despite these checklists demonstrating large progress in the knowledge of freshwater fish taxonomy of the Rokel River basin or the upper Guinea ecoregion, they no longer sufficiently define the diversity and distribution of species within the RR basin or the entire country. From the late 1990s to the present, 72 species of fish have been recognized across this area [1,3,4].
The Cyprinidae, with 13 currently recognized species, is the second dominant component of the fish fauna of the RR basin, accounting for 18.1% of the total number of species in this river. Among them, there are two regional endemic species: Leptocypris guineensis, Daget 1962 and Prolabeo batesi, Norman 1932. The remaining species are widely regarded as “amphi-Guinean”, occupying freshwater systems in the lower and upper Guinean ecoregion or even more broadly throughout West Africa [1,3]. However, the taxonomic status of some currently recognized morphology-based species, particularly widespread species, of the upper Guinea ecoregion needs to be scrutinized using an integrated taxonomy approach.
This study represents the first step in this process, being the first molecular assessment of the RR basin to determine the level of genetic distinctiveness of these populations to ascertain if impending concerns with their existing taxonomic status can be identified.
2. Material and Methods
2.1. Study Area
The Rokel River basin drains the uplands of northern Sierra Leone at altitudes of 300–400 m, where the coastal plain borders the Guinea Plateau, and drains about 300 km into the Atlantic through the estuarine Sierra Leone River (Figure 1). Specimens were caught from different portions within the RR basin (Figure 1) by monofilament gill nets with mesh sizes of 10–35 mm, seine, cast net, and fish traps. Samples were preserved in 95% ethanol and other specimens were fixed in 10% formalin and stored in 10%, 50%, to 70% ethanol for prolonged storage. Preliminary species’ identification was based on morphology. Samples were assigned to five genera: Enteromius spp., Labeo spp., Labeobarbus spp., Raiamas spp., and Prolabeo batesi.
All collected specimens are deposited in the Museum of Aquatic Organism at the Institute of Hydrobiology (IHB), Chinese Academy of Sciences, Wuhan city, Hubei Province. The “cf.” was used in this study to describe species whose identity was uncertain. The “aff” was used in this study to describe species whose identity was uncertain, and it was classified as inquirenda to indicate the need for further taxonomic review to confirm its taxonomic validity, following the definition of [18].
Total DNA was extracted from ethanol-stored fin tissue using a TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China). A single fragment of the mitochondrial cytochrome b (Cytb) gene was utilized for phylogenetic analysis. This gene was amplified by a PCR reaction, using universal fish DNA primers LA-CYTB (Yang)/HA-CYTB (Yang), following published protocols [19,20]. All sequences generated from this study were deposited in GenBank.
2.2. Data Analysis
The DNA analysis included published cytochrome b (Cytb) data from other cyprinids in the area [9,20] and newly amplified Cytb gene sequences markers. Sequences were cleaned, aligned, and trimmed to equal lengths utilizing the following programs: MAFFT [21] and AliView [22]. Amino acid sequences were inspected to ascertain that there were no stop codons present. Datasets were examined for redundancy and saturation using DAMBE v7.0.35 [23]. Individual haplotypes were determined by DnaSP v5 [24].
The suitable models of sequence evolution for each dataset were selected under Akaike’s Information Criterion (AIC) [25]. RAxML v8.2 [26] was utilized for Maximum Likelihood (ML) analysis, with model substitution rates from Modeltest applied [27]. MRBAYES 3.1.1 [28] was utilized for Bayesian Inference (BI) analysis with the best-fit model for each partition selected by Partionfinder2 in PhyloSuite [21]. Minimum and maximum pairwise differences were estimated in MEGA 7 [29] using the Kimura 2-parameter model, and partial deletion of missing data (90% site coverage cut-off). Species, sampling locality, voucher numbers, and GenBank accession numbers used for phylogenetic analysis are listed in Table 1.
2.3. Phylogenetic Analysis
An alignment of partial Cytb (1052 bp) from 101 specimens was included in the analysis. This included 22 specimens from Enteromius, 3 specimens of Labeo, 3 specimens of Labeobarbus, 2 specimens from Raiamas, and 2 specimens of Prolabeo from the RR basin, and published congeneric species from West Africa or Africa.
Phylogenetic relationships among morphospecies between groups were inferred using IQ-Tree 2.1.2 [21]. The analysis for each group had two replicate searches, six million generations, with four Markov chains. Trees were sampled every 1000 generations to obtain 10,000 sampled trees. We discarded 25% of the sampled trees as burn-in, and the remainder were used to estimate the consensus tree and Bayesian posterior probabilities (PP). The average standard deviation of split frequencies and the potential scale reduction factor were estimated. The values depicted are a posteriori probability for BI/ML. Posteriori probability was based on nodal support for BI/ML trees. Model-corrected genetic distances between unique lineages recognized for each genus were estimated using MEGA7 [29]. To discover the probable taxonomic individuality of the genetic lineages that will be unveiled from the RR basin, sequences of topotypes (i.e., published sequences in the upper Guinea Province of West/West-Central Africa of formerly described species) were included in the analysis. Mean distances within and between species were computed for two of the better-performing versions of each species delimitation method described below: ASAP (recursive partitioning) and bPTP (maximum likelihood).
2.4. Species Delineation
The ASAP analysis using the online server (https://bioinfo.mnhn.fr/abi/public/asap (accessed on 15 December 2021)) was performed to divide the group into hypothetical species based on the genetic distance, which can be observed whenever the divergence among populations that belonged to the same species is smaller than the divergence among populations from different species. The coalescent clustering-based method (bPTP) was performed using the online server (https://species.h-its.org/ (accessed on 15 December 2021)) and the Bayesian Inference trees from MrBayes 3.1.1 [28]. We ran bPTP analyses for 500,000 MCMC generations, with a thinning of 500 and a burn-in of 0.1. Convergence of the MCMC chain was assessed as recommended by Zhang et al. [21,27]. Outgroups were pruned before conducting bPTP analyses to avoid bias that may arise if some of the outgroup taxa were too distantly related to the ingroup taxa. The phylogenetic trees were visualized and edited in FigTree v.1.4.3 (Institute for Evolutionary Biology | Centre for Infection, Immunity & Evolution Ashworth Laboratories, University of Edinburgh, Edinburgh, EH9 3FL, UK), Adobe Illustrator CS6, and Adobe Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA).
3. Results
Highly supported phylogenies inferred by both BI and ML analysis discovered 11 distinct populations within cyprinids from the RR (Figure 2 and Figure 3).
Phylogenetic analysis of 8 unique haplotypes of Labeo (3), Labeobarbus (3), and Raiamas (2) and 29 published sequences of the same gene from closely allied West and West-Central African species of these 3 genera yielded identical tree topology from ML and BI methods, with most branches receiving strong support. ASAP and bPTP analyses detected six putative species for cyprinids of the RR basin (Figure 3).
Samples identified as Labeobarbus, Labeo, and Raiamas species from the RR basin of Sierra Leone were clustered within four clades (A–E) in the resulting molecular phylogenetic tree (Figure 3). Two species delineation analyses recognized six putative species: L. coubie, L. parvus, L. wurtzi, L. sacratus, R. steindachneri, and R. scarciensis.
Specimens from the RR basin described here as L. wurtzi clustered within Clade A, where it grouped with published sequences (AF287445 and AF180868) from the species; in addition, sequences from L. sacratus (Clade B) of the RR basin also clustered with published sequences (AF287448 and AF180864) of the species from Guinea (type locality). The genetic divergence between these two species was 5.4% and their intraspecific genetic divergence was 0.6% and 1.3%, respectively (Table 2).
Labeo aff. parvus of the RR basin was clustered within Clade C, where it formed a strongly supported lineage, being a sister to L. parvus of the Nile River basin (type locality), and their genetic distance was 7.1%. Their interspecific genetic distance was 8.0% with L. parvus from this river basin.
Sequences of Labeo aff. coubie were clustered within Clade D, which was a sister to L. coubie, a widespread species of West-Central and East Africa (GenBank number: AP012149.1; JX074261.1). Their genetic distance was 5.7%. The paired species nested with L. cyclorhynchus of the Ogooué River basin (type locality). The genetic distance of L. aff. coubie was 9.7% with L. cyclorhynchus (Table 2).
Sequences of Raiamas from the RR basin were nested within Clade E. The sequence from the species under the name of R. cf. steindachneri was clustered with R. scarciensis from the same river basin as the GenBank-retrieved sequence (AP012113.1) of R. steindachneri, without precise sampling location. The genetic divergence between species Raiamas from the RR basin was 21.8% (Table 2).
The data matrix of the Cytb gene for 11 haplotypes from species of Enteromius (9) and Prolabeo (2) from the RR and 56 published sequences from closely allied species of Enteromius from West and West-Central Africa (including East Africa) were subjected to phylogenetic analyses, with Paracanthocobitis zonalternans and P. mockenziei from Asia used as the outgroup (Figure 3). Both ML and BI methods yielded identical tree topology, with most branches receiving strong support, and both ASAP and bPTP species delineation analyses recognized five putative species for cyprinids of the RR basin.
Species of Prolabeo and Enteromius from the RR basin were clustered within five clades (A–E) in the BI and ML trees (Figure 3). The putative species was represented in Clade A by samples of P. batesi from the RR basin of Sierra Leone (type locality). Its sister species was E. trispilos from the Mia River at Bourata Village (type locality). Their genetic divergence was 16.5% (Table 3).
Among four putative species delineated within clade B, one was represented by samples from E. aff. macrops of the RR basin. Its sister species was E. macrops s.str., from type locality: Kolenté or Tinkisso River. The genetic distance between these paired species was 2.4%. These two populations had an interspecific genetic distance of 2.7% and 3.5% with the other two E. aff. macrops 1 and 2 (Figure 3) putative species formed, respectively, by GenBank-retrieved sequences (MF135212, MF135200, MF135205, MF135210) and (MF135202, MF135202, MF135211, MF135206, MF135204, AF180832) from samples of Forécariah, Koumba, and Tinkisso River basins, and those from the Konkouré and Doulou River basins (Guinea; type localities).
Samples of E. foutensis from the RR basin represented a putative species (E. aff. foutensis), one of four putative species delineated within Clade C. Its sister species was formed by samples of E. foutensis s.str., and also one sample formerly identified as E. bigornei, from the Little Scarcies River basin (type locality). The interspecific genetic distance between E. foutensis s.str. and E. aff. foutensis was 3.9%. The paired species were sisters to the paired species (E. aff. foutensis of the Konkouré River basin and the Gambie/Senegal River basins), with which E. aff. foutensis of the RR basin had an interspecific genetic distance of 5.1% and 6.8%, respectively. Nested within E. aff. foutensis of the Gambie/Senegal River basins were GenBank-retrieved sequences formerly misidentified as E. punctitaeniatus (MF135199) and E. ablabes (MF135227 and MF135228). Their interspecific genetic distance ranged from 0.01% to 0.09% (Table 3).
Only two putative species were delineated within Clade D. One was formed by samples from E. aff. ablabes of the RR basin. It was sister to the other represented by the sample from E. anema s.str. (type locality: Nile basin). The paired species clustered with E. profundus from Lake Victoria into a lineage, being sister to E. cf. guildi from the Bafing River (at Sokotoro, East of Timbo). The basal placement of Clade D was occupied by E. ablabes s.str. from the Agnebi River of the Ivory Coast (type locality). The interspecific genetic distances of E. aff. ablabes in the RR basin were 9.8% with E. anema, 10.1% with E. profundus, 14.4% with E. cf. guildi, and 11.9% with E. ablabes s.str. Samples from E. aff. liberiensis of the RR basin represented a putative species of Clade E (Figure 3), where it stood out as the sister species of E. cadenati, endemic to the Pampana/Jong or Taia River basin of Sierra Leone, with their interspecific genetic distance of 4.6%. The true E. liberiensis of the Safa-Khoure River was sister to E. tiekoroi from Kolenté and Little Scarcies basins. Enteromius liberiensis had a distinct genetic distance of 13.3% with E. aff. liberiensis.
4. Discussion
4.1. Misidentification and Unrecognized Diversity
This study exhibits, to large extent, the misidentification of the currently recognized cyprinids and thus the existence of unappreciated diversity of the RR basin. This river harbors ten species of four genera within the Cyprinidae: Enteromius (four), Labeo (two), Labeobarbus (two), and Raiamas (two) [1,3,4]. Our samples of cyprinid fish collected from the RR basin were initially recognized as 11 morphospecies. While E. leonensis eluded capture, two other newly recorded species of the river were caught: Enteromius foutensis and Prolabeo batesi. The Cytb gene-based phylogenetic analyses utilizing BI and ML approaches recovered 11 distinct lineages, and species delineation analyses also recognized them as 11 putative species (Figure 2 and Figure 3). The identification of four species (Labeobarbus wurtzi, L. sacratus, Prolabeo batesi, and Raiamas scarciensis) is confirmed. Raiamas steindachneri is tentatively considered as of taxonomic uncertainty. However, two species of Labeo from the RR basin are demonstrated to be currently misidentified, and so are four species of Enteromius.
Raiamas steindachneri and Raiamas scarciensis, typical species of the upper Guinean ecoregion, occur in the coastal rivers of Guinea, Liberia, and Sierra Leone. The species herein recognized from the RR basin formed two distinct lineages that were delineated as a putative species (Figure 3). These species had a distinct genetic distance of 23.3%, greater than the threshold value (2%), which is the cut-off value commonly utilized to denote intraspecific variation [29,30].
Two currently recognized species of L. coubie and L. parvus from the RR basin are misidentified. Samples initially referred to as each of both formed a distinct lineage, which was distantly allied to the lineage made up of its topotypical samples in the molecular phylogenetic tree (Figure 2). There is a distinct genetic distance between L. aff. coubie (RR basin) and L. coubie s.str. (6.8%) and L. aff. parvus (RR basin) and L. parvus s.str. (7.1%). Perhaps these two species, i.e., L. aff. coubie and L. aff. parvus, represent species of Labeo curriei, Fowler 1919 and L. obscurus, Pellegrin 1908, originally described from this area [4]. The taxonomy of these species is not clear, and more specimens and sampling areas are required.
The type locality of E. macrops is the Little Scarcies River basin of Sierra Leone. Samples initially identified as this species from the RR basin formed a distinct lineage that was delineated as a putative species (E. aff. macrops) (Figure 3). Its genetic distance with E. macrops s.str. was 2.7%, slightly greater than the 2% threshold (Table 3). Hence, more specimens and the use of the integrative method are essential to determine its taxonomic status. The same holds for the samples initially identified as E. foutensis from the river. These samples represent a distinct species (E. aff. foutensis) due to its monophyletic nature recovered in the Cytb gene-based tree (Figure 3) and its significant genetic distance with E. foutensis s.str. (3.9%) from the Little Scarcies of Sierra Leone (Table 3).
Samples initially identified as either E. ablabes or E. liberiensis from the RR basin formed a distinct lineage distantly allied to the lineage made up of its topotypical samples, and two distinct lineages were delineated as two putative species: E. aff. ablabes and E. aff. liberiensis (Figure 3). There was a significant genetic distance between E. aff. ablabes of the RR basin and E. ablabes of the Agnebi River basin (type locality) of the Ivory Coast (10.1%), and between E. aff. liberiensis and E. liberiensis from the Safa-Khoure River basin (type locality) of Guinea (13.3%). The genetic distance of E. aff. ablabes and E. aff. liberiensis, respectively, with its sister species E. aff. anema from the Blue Nile River basin and E. cadenati from the Pampana/Jong River basin of Sierra Leone, was 9.8% and 4.6%. Based on these molecular data, it can be concluded that Enteromius aff. ablabes and Enteromius aff. liberiensis of the RR basin belong to two unnamed species. This study also highlights a need to put the current morphology-based species of Enteromius under molecular scrutiny, as indicated in the previous investigation. Type specimens of two new species, E. alberti and E. mimus, were previously considered conspecific, respectively, with E. perince and E. stigmatopygus [31]. Decru et al. [13] also reported deep divergence among four morphology-based species of Enteromius from the northeastern part of the Congo River basin, and their reported genetic divergence was greater than 5% and even up to 20% between lineages of morphologically similar specimens, clearly surpassing the 2% threshold. Taxonomic revisions of fish in the upper Guinean ecoregion suggest the likely discovery of new species in the Fouta Djallon Highlands. Integrative analyses applied to the African mountain catfish (Amphilius spp.) of Fouta Djallon Mountain resulted in the discovery of at least nine new species of Amphilius and small barbs [10,11,12]. Only 11 morphospecies of cyprinid fish from the RR basin were investigated here in a molecular phylogenetic context, but seven of them were shown to be misidentified or of taxonomic uncertainty. If this level of scrutiny is extrapolated to all morphospecies collected from the RR basin, it is likely that new species and even more endemic species may be discovered.
4.2. Genetic Placement of Prolabeo Batesi
Despite the mouth structure and body shape, Prolabeo batesi is considered a monotypic genus (i.e., distinct from Enteromius and Labeo) based on morphological characteristics [1,3,4]. Several characters placed this genus close to L. wurtzi [3,4]. This genus has never been compared with large taxa in a molecular phylogenetic-based approach. This is the first molecular analysis of the phylogenetic relationship of Prolabeo with others. It is revealed that Prolabeo was not recovered as a distinct lineage; instead, it was resolved as the sister species of Enteromius trispilos (see Figure 3). According to Paugy et al. [4], E. trispilos differs from all other three-spotted barbs by its elongated body, a character very similar to the genus Prolabeo. We therefore suggested that E. trispilos should be assigned to the genus Prolabeo.
4.3. Biogeographic Implication
Biogeographic and phylogenetic relationships of cyprinids from the RR basin with their sister species suggest that the RR had complicated historical inter-basin connections with other nearby river basins of the upper Guinean and Nilo-Sudan ecoregions, or even with the Congo basin and Lower Guinean ecoregion [8,32].
The past connection of the RR basin with some nearby river basins of the upper Guinean ecoregion is suggested by several paired species. The sister pair E. aff. macrops and E. macrops have an allopatric distribution in the RR basin and the Forécariah basin, and the same pattern is also repeated for the sister pair E. aff. foutensis of the RR basin and E. foutensis of the Little Scarcies River basin. The paired species E. aff. liberiensis and E. cadenati exhibit a disjunct distribution in the RR basin and the Pampana/Jong River basin (Figure 3). All these paired species are usually found in the upper reaches of rivers [10]. It is suggested here that headwater capture may have been the cause of inter-basin transfer, in which one stream cuts through the watershed separating the two basins, allowing the stream that clears the cut to get the stream from the second basin. Fish and other biota from the captured stream are thereby introduced into the stream that makes the capture, establishing new subdivided populations. The watershed of the stream that makes the capture is increased in the area only by the part of the captured watershed above the cut [7,8,9]. These events possibly occurred during interglacial phases when the forests would have expanded as the weather became wetter. This climatic undulation and the concomitant expansion/contraction of forest cover were probably the main factors leading to the diversity and geodispersal patterns of the biota seen today [5,8,32,33].
In the BI and ML trees (Figure 3), E. aff. ablabes from the upper reaches of the RR basin had a closer relationship with E. anema from the upper Niger River basin of the Nilo-Sudan ecoregion. This allopatric distribution is also displayed by the two sister pairs L. aff. parvus and L. aff. coubie from the RR basin and L. parvus and L. coubie from the upper Niger River basin of the Nilo-Sudan ecoregion. The tectonic events during the Miocene period modified the hydrographic system, wherein populations from the Nilo-Sudan were separated by the uplift of the Fouta Djallon. Hence, this event can be a plausible explanation for the vicarious forms of their ancestral population [34]. The later formation of the watershed between them was the key driving force for their differentiation into distinct species to form a repeated disjunct distribution. More research on inter- and intra-specific relationships between different taxa occurring in neighboring rivers or ecoregions could help in unraveling the complex biogeographic patterns of this area.
4.4. Endemism and Conservation Implications
The revelation of unrecognized diversity of cyprinid fish from the RR basin in this study suggests important implications for the conservation of biodiversity. Inaccurate taxonomy on purely morphological and/or molecular grounds only leads to an underestimation of species richness and endemism, which can misdirect conservation efforts [13,16,34]. However, our DNA molecular approach used in this study is not enough to identify species for conservation efforts, and the team is working on a morphological approach. An integrative taxonomic revision of cyprinids from the RR basin in the future is likely to confirm that four putative species (L. aff. parvus, L. aff. coubie, E. aff. ablabes, and E. aff. macrops) delineated here are endemic to the upper Guinean ecoregion/Sierra Leone or RR basin, and thus of particular conservation concern. Unfortunately, these putative species are currently misidentified as four amphi-Guinean or Pan-African species (L. parvus, L. coubie, E. ablabes, and E. macrops), which are so far assessed as Least Concern (LC) [35]. Subsequently, freshwater fish species from this area are not listed as target species for conservation planning and priority [17], probably due to the underestimated level of endemism and the overestimated level of widely distributed populations. This ecoregion is presently not listed among the priority freshwater Key Biodiversity Areas within the upper Guinean Biodiversity Hotspot [17]. Unveiling this undetected diversity of the RR basin fish might warrant reconsideration for conservation priorities.
Two species of Enteromius (E. foutensis and E. liberiensis), endemic to the upper Guinean ecoregion, are currently listed as Endangered (EN) [16,35]. Both species are conventionally defined based on morphological characteristics alone, a cryptica species complex. E. foutensis consists of at least three putative species [10], and E. foutensis and E. liberiensis are shown here to contain four and two putative species, respectively. These results permit an updated assessment of the conservation status of E. aff. foutensis and E. aff. liberiensis of the RR basin. The two putative species have a narrower distribution than previously identified morphospecies. The extent to which both are under threat is more severe than presently supposed. The same is the case for the other four putative species (L. aff. parvus, L. aff. coubie, E. aff. ablabes, and E. aff. macrops) of the RR basin. This unappreciated diversity merits consideration in conservation planning.
Considering that, six putative cyprinid species delineated here from the RR basin remain unnamed, efforts to catalog and assess the fish diversity of this river need to be prioritized as the first step for conservation of fish species diversity. The high level of endemism observed today is mainly attributed to the isolation of the RR basin from neighboring river basins in Sierra Leone or the upper Guinean ecoregion and its geographic position [1,2]. This river also holds a wealth of mineral resources and high hydropower potential. Fish species of the RR basin, particularly the upper reaches upstream of the Bumbuna dam, are susceptible to natural factors and anthropogenic disturbances. The most common threats observed in this area are uncontrolled timber logging, gold-mining activities, the expansion of the Bumbuna dam, and uncontrolled fishing activities [1]. These activities all bring about habitat degradation triggered by deforestation and pollution. These modifications are deleterious to native species, including the six putative species delineated herein. Thus, these putative species may go extinct before they are officially described. Species identification is essential for species-based conservation and management [34,36,37]. It is an urgent need to gain a better understanding of the biodiversity of the RR basin and beyond to prioritize biodiversity conservation, particularly that under threat from ongoing deforestation.
Author Contributions
U.C.K., E.Z., J.S., C.C.N. and C.L. contributed to the design, data analysis, writing, and general discussion of the taxonomic part; U.C.K. and C.L. took lead on the sampling and general laboratory work. All authors have read and agreed to the published version of the manuscript.
Funding
This research is supported by a grant from the Sino-Africa Joint Research Centre (Nos. SAJC201320 and SAJC201612). The authors assert that the funding body had no role in the design of the study, in the collection, analysis, and interpretation of data, or in writing the manuscript.
Institutional Review Board Statement
Specimens utilized for this study were sampled in accordance with the Chinese Laboratory Animal Welfare and Ethics animal welfare laws (GB/T 35892–2018).
Data Availability Statement
The nucleotide sequence data that support the findings of this study are openly accessible in GenBank of NCBI at https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/websub/?form=history&tool=genbank under the accession no. MW660579-601; MZ013919-22.
Acknowledgments
The authors thank Kanu, A.R. and Brima, S.U. (deceased) for their support in collecting samples throughout our stay in Sierra Leone. We appreciate Ellie, E. and Jalloh, K., from the Ministry of Fishery and Marine Resources for their help to facilitate sampling and export permits. Our thanks go to An, C.T., Chen, X., Melaku, Y.A., Guo, D.M. Deng, S.Q., Shao, W.H. and Nguyen, D.T. (IHB) for their help with the molecular technique experiment, and Liu, Y. (IHB) for making figures using Adobe. The authors thank the reviewers and editors of this article for their very constructive comments and suggestions.
Conflicts of Interest
The authors declare no conflict of interest.
Abbreviations
The following abbreviations were used in this manuscript:
| RR | Rokel River basin |
| IHB-CAS | Institute of Hydrobiology, Chinese Academy of Sciences |
| ASAP | Assemble Species by Automatic Partitioning |
| bPTP | Poisson Tree Processes |
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Figure 1.
Distributions of sampled specimens of fish species from the Rokel River basin. The green dots show the sampling site, and the black dot is the position of the Bumbuna hydro-dam.
Figure 1.
Distributions of sampled specimens of fish species from the Rokel River basin. The green dots show the sampling site, and the black dot is the position of the Bumbuna hydro-dam.
Figure 2.
Phylogeny of species of Labeo, Labeobarbus, and Raiamas inferred from partial cytochrome b. Branch support for each node is shown from Bayesian Inference (Lineages A–E). The right vertical bars indicate partitions and final MOTUs from ASAP and bPTP. Note: Species marked with asterisks are considered ambiguous or possible misidentification. Species in red are those from the RR basin.
Figure 2.
Phylogeny of species of Labeo, Labeobarbus, and Raiamas inferred from partial cytochrome b. Branch support for each node is shown from Bayesian Inference (Lineages A–E). The right vertical bars indicate partitions and final MOTUs from ASAP and bPTP. Note: Species marked with asterisks are considered ambiguous or possible misidentification. Species in red are those from the RR basin.
Figure 3.
Phylogeny of species of Enteromius and Prolabeo inferred from partial cytochrome b. Branch support for each node is shown from Bayesian Inference (Lineages A–E). The right vertical bars indicate partitions and final MOTUs from ASAP and bPTP. Note: Species marked with asterisks are considered ambiguous or possible misidentification. Species in red are those from the RR basin.
Figure 3.
Phylogeny of species of Enteromius and Prolabeo inferred from partial cytochrome b. Branch support for each node is shown from Bayesian Inference (Lineages A–E). The right vertical bars indicate partitions and final MOTUs from ASAP and bPTP. Note: Species marked with asterisks are considered ambiguous or possible misidentification. Species in red are those from the RR basin.
Table 1.
Sequences used for this study and published congeneric sequences from West Africa or Africa ecoregions.
Table 1.
Sequences used for this study and published congeneric sequences from West Africa or Africa ecoregions.
| Species | Specimens ID | Countries | Locality/River | GenBank No. | Year Collected |
|---|---|---|---|---|---|
| Enteromius anema | AUF5493 | Guinea | Bafing | MF135226.1 | Hayes and Armbruster 2017 |
| Enteromius anema | AUF5494 | Guinea | Bafing | MF135225.1 | |
| Enteromius ablabes | Unknown | Ivory Coast | Agnebi | AF180835.1 | Tsigenopoulos et al., 1999 |
| Enteromius macrops | AUF 5524 | Guinea | Forécariah | MF135212.1 | |
| Enteromius macrops | Gui048 | Guinea | Forécariah | MF135201.1 | |
| Enteromius macrops | Gui0237 | Guinea | Forécariah | MF135200.1 | |
| Enteromius macrops | AUF5481 | Guinea | Safa-Khoure | MF135210.1 | |
| Enteromius macrops | Guinea | Koumba | MF135203.1 | ||
| Enteromius macrops | Guinea | Koumba | MF135202.1 | ||
| Enteromius macrops | AUF5454 | Guinea | Tinkisso | MF135204.1 | |
| Enteromius macrops | Guinea | Tinkisso | MF135205.1 | ||
| Enteromius macrops | Guinea | Kolenté | MF135208.1 | ||
| Enteromius macrops | AUF5476 | Guinea | Kolenté | MF135206.1 | |
| Enteromius macrops | Guinea | Kolenté | MF135209.1 | ||
| Enteromius macrops | Guinea | Kolenté | MF135207.1 | ||
| Enteromius macrops | Guinea | Konkouré | AF180832.1 | ||
| Enteromius macrops | Guinea | Doulou | MF135211.1 | ||
| Enteromius camptacanthus | Unknown | Ghana | Lake Volta | KF791270.1 | |
| Enteromius anema | Unknown | Nilo-Sudan | Blue Nile | KP712159.1 | |
| Enteromius guildi | AUF5505 | Guinea | Zie | MF135218.1 | |
| Enteromius cadenati | Unknown | Sierra Leone | Taia/Jong | AF180834.1 | |
| Enteromius cadenati | AUF5364 | Guinea | Dimmah | MF135224.1 | |
| Enteromius liberiensis | AUF5483 | Guinea | Safa-Khoure | MF135213.1 | |
| Enteromius bigornei | MNCN: 46CK | Guinea | Kaba, Kouloundela | AY004752.1 | Machordom and Doadrio 2001 |
| Enteromius aspilus | Unknown | Guinea | Konkouré | KF791275.1 | Schmidt et al., 2019 |
| Enteromius foutensis | Gui0858 | Guinea | Little Scarcies | MK329230.1 | |
| Enteromius foutensis | Gui0146 | Guinea | Little Scarcies | MK329231.1 | |
| Enteromius foutensis | Gui0167 | Guinea | Little Scarcies | MF135220.1 | |
| Enteromius foutensis | Gui0168 | Guinea | Little Scarcies | MF135219.1 | |
| Enteromius foutensis | Gui3435 | Guinea | Konkouré | MK329229.1 | |
| Enteromius foutensis | Gui3494 | Guinea | Konkouré | MK329228.1 | |
| Enteromius foutensis | Gui0145 | Guinea | Konkouré | MK329227.1 | |
| Enteromius foutensis | Gui_0206 | Guinea | Konkouré | MK329233.1 | |
| Enteromius foutensis | Gui0146 | Guinea | Konkouré | MK329226.1 | |
| Enteromius foutensis | Gui 0146 | Guinea | Konkouré | MK329225.1 | |
| Enteromius foutensis | Gui_0204 | Guinea | Konkouré | MK329232.1 | |
| Enteromius foutensis | Gui 0167 | Guinea | Konkouré | MK329224.1 | |
| Enteromius foutensis | Gui 0018 | Senegal | Gambie/Senegali | MK329241.1 | |
| Enteromius foutensis | Gui 0133 | Senegal | Gambie/Senegali | MK329240.1 | |
| Enteromius cf. guildi | AUF5443 | Guinea | Bafing | MF135223.1 | Hayes and Armbruster 2017 |
| Enteromius ablabes | AUF5431 | Guinea | Bafing | MF135227.1 | |
| Enteromius ablabes | AUF5441 | Guinea | Bafing | MF135228.1 | |
| Enteromius trispilos | AUF5496 | Guinea | Mia | MF135193.1 | Hayes and Armbruster 2017 |
| Enteromius trispilos | AUF5498 | Guinea | Mia | MF135194 | |
| Enteromius anema | AUF5493 | Guinea | Mia | MF135225.1 | |
| Enteromius anema | AUF5494 | Guinea | Mia | MF135226.1 | |
| Enteromius huguenyi | AUF5589 | Guinea | Masseni | MF135214.1 | |
| Enteromius punctitaeniatus | AUF5610 | Guinea | Mafou | MF135199.1 | |
| Enteromius profundus | DNG_PROF2_2 | Kenya | Kisumu | MH484558.1 | Ndeda, Mateos, and Hurtado 2018 |
| Enteromius profundus | DNG_PROF4_4 | Kenya | Kisumu | MH484556.1 | |
| Enteromius callipterus | Unknown | Gabon | Loa -Loa | AP009313.1 | Saitoh 2006 |
| Enteromius callipterus | CBM-ZF-11498 | Gabon | Loa -Loa | KP712230.1 | |
| Labeobarbus sacratus | MNCN 4CK | Guinea | Tangala | AF287445.1 | Tsigenopoulos, Naran, and Berrebi 1999 |
| Enteromius tiekoroi | UAIC14166.05 | Sierra Leone | Mao | KP659410.1 | Yang et al., 2015 |
| Labeobarbus sacratus | Guinea | Tangala | AF180868.1 | Tsigenopoulos, Naran, and Berrebi 1999 | |
| Labeobarbus sacratus | AF287445.1 | ||||
| Labeobarbus wurtzi | MNCN 92CK | Guinea | Kouloundela | AF287448.1 | |
| Labeobarbus wurtzi | MNCN 91CK | Guinea | Kaba, | AF180864.1 | |
| Labeo forskalii | CU 94562 | Ethiopia | Alwero | JX074287.1 | Yang and Mayden 2012 |
| Labeo forskalii | UAIC14744.4 | Ethiopia | Alwero | FJ196833.1 | Beshera and Phillip 2019 |
| Labeobarbus cyclorhynchus | CBM ZF 11452 | AP011359.1 | Miya 2009 | ||
| Labeo forskalii | AAU:0512009 | Ethiopia | Alwero | FJ196831.1 | Tang, Getahun, and Liu 2009 |
| Labeo lukulae | DRC | Lukula | JX097084.1 | Hirt 2012 | |
| Labeo parvus | BMNH:2006.3.7.1 | Benin | bei Malauville | JX074292.1 | Yang and Mayden 2012 |
| Labeo parvus | CBM: ZF: 12695 | Ethiopia | Alwero | AP013339.1 | Beshera and Phillip 2019 |
| Labeo parvus | Ethiopia | Baro | JX074285.1 | Yang and Mayden 2012 | |
| Labeo parvus | Ethiopia | Baro | JX074286.1 | ||
| Raiamas senegalensis | Benin | Iguidi | AP010780.1 | Saitoh et al., 2008 | |
| Raiamas senegalensis | HM224332.1 | ||||
| Labeo horie | Ethiopia | Alwero | JX074288.1 | ||
| Labeo nasus | DRC | Congo Basin | AP013333.1 | Miya 2013 | |
| Labeo lineatus | AP012154.1 | ||||
| Labeo altivelis | AP013322.1 | Miya 2011 | |||
| Labeo coubie | Nigeria | Niger Basin | AP012149.1 | ||
| Labeo coubie | JX074261.1 | ||||
| Labeo altivelis | JX074228.1 | ||||
| Raiamas steindachneri | N/A | AP012113.1 | |||
| Raiamas cf. steindachneri | IHB29666 | Sierra Leone | Rokel/Seli/upper | MW660585 | This study |
| Raiamas aff. scarciensis | IHB29555 | Sierra Leone | Rokel/lower | MW660586 | This study |
| Labeo aff. coubie | IHB29688 | Sierra Leone | Rokel/Seli/upper | MW660599 | This study |
| Labeo aff. parvus | IHB29699 | Sierra Leone | Rokel/Seli/upper | MW660600 | This study |
| Labeo aff. parvus | IHB29799 | Sierra Leone | Rokel/Seli/upper | MW660601 | This study |
| Labeobarbus wurtzi | IHB29999 | Sierra Leone | Rokel/Seli/lower | MW660597 | This study |
| Labeobarbus wurtzi | IHB29999B | Sierra Leone | Rokel/Seli/lower | MW660597B | This study |
| Labeobarbus sacratus | IHB29898 | Sierra Leone | Rokel/Seli/upper | MW660598 | This study |
| Enteromius aff. foutensis | IHB29317 | Sierra Leone | Rokel/Seli/upper | MW660590 | This study |
| Enteromius aff. foutensis | IHB29318 | Sierra Leone | Rokel/Seli/upper | MW660591 | This study |
| Enteromius aff. foutensis | IHB29319 | Sierra Leone | Rokel/Seli/upper | MW660592 | This study |
| Enteromius aff. foutensis | IHB29320 | Sierra Leone | Rokel/Seli/upper | MW660593 | This study |
| Enteromius aff. foutensis | IHB29444 | Sierra Leone | Rokel/Seli/upper | MW660594 | This study |
| Enteromius aff. liberiensis | IHB29241 | Sierra Leone | Rokel/Seli/upper | MW660587 | This study |
| Enteromius aff. liberiensis | IHB29362 | Sierra Leone | Rokel/Seli/upper | MW660589 | This study |
| Enteromius aff. liberiensis | IHB29242 | Sierra Leone | Rokel/Seli/upper | MW660588 | This study |
| Enteromius aff. macrops | IHB29355 | Sierra Leone | Rokel/Seli/upper | MW660595 | This study |
| Enteromius aff. macrops | IHB29610 | Sierra Leone | Rokel/Seli/upper | MW660596 | This study |
| Enteromius aff. ablabes | IHB29544 | Sierra Leone | Rokel/Seli/upper | MZ013921 | This study |
| Enteromius aff. ablabes | IHB29545 | Sierra Leone | Rokel/Seli/upper | MZ013922 | This study |
| Prolabeo batesi | IHB29377 | Sierra Leone | Rokel/lower | MZ013919 | This study |
| Prolabeo batesi | IHB29399 | Sierra Leone | Rokel/lower | MZ013920 | This study |
| Paracanthocobitis zonalternans | Asia | MK608087.1 | Slechtova and Dvorak 2019 | ||
| Paracanthocobitis mockenziei | MK608121.1 |
Table 2.
Mitochondrial Cytb genetic distances between lineages and species of the Labeo spp., Labeobarbus spp., and Raiamas spp., from the RR basin, Sierra Leone, West Africa.
Table 2.
Mitochondrial Cytb genetic distances between lineages and species of the Labeo spp., Labeobarbus spp., and Raiamas spp., from the RR basin, Sierra Leone, West Africa.
| No. | Species | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Labeobarbus wurtzi | ||||||||||||
| 2 | L. sacratus | 0.054 | |||||||||||
| 3 | Labeo forskallii | 0.182 | 0.157 | ||||||||||
| 4 | L aff. parvus | 0.180 | 0.159 | 0.080 | |||||||||
| 5 | L. parvus | 0.177 | 0.157 | 0.072 | 0.071 | ||||||||
| 6 | L. cyclorhynchus | 0.146 | 0.147 | 0.135 | 0.135 | 0.113 | |||||||
| 7 | L. longipinnis | 0.147 | 0.154 | 0.142 | 0.141 | 0.137 | 0.098 | ||||||
| 8 | L. aff. coubie | 0.161 | 0.168 | 0.135 | 0.143 | 0.130 | 0.097 | 0.068 | |||||
| 9 | L. coubie | 0.147 | 0.148 | 0.121 | 0.131 | 0.118 | 0.082 | 0.065 | 0.057 | ||||
| 10 | R. aff. steindachneri | 0.283 | 0.268 | 0.233 | 0.245 | 0.238 | 0.246 | 0.260 | 0.257 | 0.251 | |||
| 11 | R. steindachneri | 0.275 | 0.269 | 0.236 | 0.245 | 0.241 | 0.253 | 0.262 | 0.259 | 0.258 | 0.037 | ||
| 12 | R. scarciensis | 0.305 | 0.289 | 0.274 | 0.263 | 0.276 | 0.294 | 0.283 | 0.297 | 0.290 | 0.218 | 0.233 | |
| 13 | R. senegalensis | 0.292 | 0.284 | 0.268 | 0.259 | 0.263 | 0.249 | 0.281 | 0.260 | 0.260 | 0.216 | 0.229 | 0.204 |
Table 3.
Mitochondrial Cytb genetic distances between lineages and species of the Enteromius Species groups from the RR basin, Sierra Leone, West Africa.
Table 3.
Mitochondrial Cytb genetic distances between lineages and species of the Enteromius Species groups from the RR basin, Sierra Leone, West Africa.
| No. | Species | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Prolabeo batesi | ||||||||||||||||||||||
| 2 | Enteromius trispilos | 0.155 | |||||||||||||||||||||
| 3 | Enteromius cadenati | 0.161 | 0.157 | ||||||||||||||||||||
| 4 | Enteromius aff. cadenati | 0.183 | 0.147 | 0.165 | |||||||||||||||||||
| 5 | Enteromius aff. liberiensis RR | 0.178 | 0.154 | 0.182 | 0.046 | ||||||||||||||||||
| 6 | Enteromius liberiensis | 0.149 | 0.118 | 0.159 | 0.129 | 0.133 | |||||||||||||||||
| 7 | Enteromius tiekoroi | 0.161 | 0.132 | 0.171 | 0.145 | 0.160 | 0.090 | ||||||||||||||||
| 8 | Enteromius macrops s.str. | 0.156 | 0.147 | 0.166 | 0.173 | 0.179 | 0.130 | 0.134 | |||||||||||||||
| 9 | Enteromius aff. macrops1 | 0.150 | 0.137 | 0.162 | 0.158 | 0.166 | 0.119 | 0.132 | 0.035 | ||||||||||||||
| 10 | Enteromius aff. macrops2 | 0.148 | 0.143 | 0.159 | 0.157 | 0.162 | 0.120 | 0.138 | 0.027 | 0.027 | |||||||||||||
| 11 | Enteromius aff. macrops RR | 0.148 | 0.145 | 0.169 | 0.169 | 0.175 | 0.121 | 0.134 | 0.027 | 0.029 | 0.024 | ||||||||||||
| 12 | Enteromius anema | 0.150 | 0.134 | 0.170 | 0.145 | 0.163 | 0.131 | 0.139 | 0.132 | 0.128 | 0.136 | 0.139 | |||||||||||
| 13 | Enteromius guildi | 0.155 | 0.138 | 0.171 | 0.150 | 0.169 | 0.135 | 0.139 | 0.135 | 0.129 | 0.142 | 0.142 | 0.006 | ||||||||||
| 14 | Enteromius aff. foutensis RR | 0.153 | 0.148 | 0.169 | 0.139 | 0.156 | 0.132 | 0.123 | 0.126 | 0.124 | 0.123 | 0.123 | 0.101 | 0.104 | |||||||||
| 15 | Enteromius foutensis s.str. | 0.151 | 0.144 | 0.167 | 0.137 | 0.141 | 0.128 | 0.134 | 0.121 | 0.118 | 0.117 | 0.122 | 0.105 | 0.109 | 0.039 | ||||||||
| 16 | Enteromius aff. foutensis1 | 0.162 | 0.148 | 0.167 | 0.143 | 0.154 | 0.133 | 0.126 | 0.122 | 0.115 | 0.117 | 0.124 | 0.087 | 0.089 | 0.068 | 0.068 | |||||||
| 17 | Enteromius aff. foutensis2 | 0.155 | 0.149 | 0.166 | 0.157 | 0.164 | 0.137 | 0.125 | 0.120 | 0.118 | 0.117 | 0.121 | 0.087 | 0.089 | 0.083 | 0.080 | 0.051 | ||||||
| 18 | Enteromius ablabes | 0.160 | 0.151 | 0.163 | 0.161 | 0.165 | 0.137 | 0.125 | 0.121 | 0.120 | 0.120 | 0.124 | 0.094 | 0.097 | 0.087 | 0.080 | 0.052 | 0.009 | |||||
| 19 | Enteromius punctitaeniatus | 0.162 | 0.153 | 0.165 | 0.162 | 0.165 | 0.137 | 0.126 | 0.122 | 0.121 | 0.122 | 0.124 | 0.096 | 0.098 | 0.087 | 0.081 | 0.054 | 0.011 | 0.001 | ||||
| 20 | Enteromius aff. ablabes1 | 0.144 | 0.137 | 0.134 | 0.164 | 0.161 | 0.134 | 0.145 | 0.129 | 0.128 | 0.123 | 0.129 | 0.137 | 0.140 | 0.118 | 0.125 | 0.118 | 0.112 | 0.112 | 0.113 | |||
| 21 | Enteromius profundus | 0.147 | 0.139 | 0.162 | 0.170 | 0.190 | 0.131 | 0.134 | 0.135 | 0.131 | 0.129 | 0.133 | 0.127 | 0.127 | 0.128 | 0.128 | 0.130 | 0.118 | 0.125 | 0.127 | 0.138 | ||
| 22 | Enteromius aff. anema | 0.147 | 0.149 | 0.167 | 0.151 | 0.168 | 0.128 | 0.132 | 0.139 | 0.124 | 0.135 | 0.135 | 0.133 | 0.134 | 0.116 | 0.128 | 0.120 | 0.108 | 0.113 | 0.115 | 0.126 | 0.116 | |
| 23 | Enteromius aff. ablabes RR | 0.142 | 0.137 | 0.156 | 0.133 | 0.139 | 0.106 | 0.120 | 0.135 | 0.130 | 0.137 | 0.139 | 0.118 | 0.120 | 0.118 | 0.113 | 0.116 | 0.106 | 0.104 | 0.105 | 0.119 | 0.101 | 0.098 |
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Kanu, U.C.; Liang, C.; Nwafor, C.C.; Shen, J.; Zhang, E. Phylogenetic Analyses of Cyprinid Species from the Rokel River Basin of Sierra Leone, West Africa: Taxonomic, Biogeographic, and Conservation Implications. Diversity 2022, 14, 299. https://0-doi-org.brum.beds.ac.uk/10.3390/d14040299
AMA Style
Kanu UC, Liang C, Nwafor CC, Shen J, Zhang E. Phylogenetic Analyses of Cyprinid Species from the Rokel River Basin of Sierra Leone, West Africa: Taxonomic, Biogeographic, and Conservation Implications. Diversity. 2022; 14(4):299. https://0-doi-org.brum.beds.ac.uk/10.3390/d14040299
Chicago/Turabian StyleKanu, Unisa Conteh, Cao Liang, Chinedu Charles Nwafor, Jianzhong Shen, and E Zhang. 2022. "Phylogenetic Analyses of Cyprinid Species from the Rokel River Basin of Sierra Leone, West Africa: Taxonomic, Biogeographic, and Conservation Implications" Diversity 14, no. 4: 299. https://0-doi-org.brum.beds.ac.uk/10.3390/d14040299
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