﻿Hidden in the riffles: A new suckermouth catfish (Mochokidae, Chiloglanis) from the middle Zambezi River system, Zimbabwe

﻿Abstract The recent surge in the discovery of hidden diversity within rheophilic taxa, particularly in West and East Africa, prompted a closer examination of the extent to which the current taxonomy may obscure the diversity of riffle-dwelling suckermouth catfishes in the genus Chiloglanis in southern Africa. Currently, the region comprises eight valid species within this genus. Seven of them have relatively narrow geographic distribution ranges except for C.neumanni, which is considered to be widely distributed, occurring from the Buzi River system in the south, and its northern limit being the eastward draining river systems in Tanzania. Recent surveys of the middle Zambezi River system revealed Chiloglanis specimens that were distinguishable from the known species of the genus from southern Africa. Integration of molecular and morphological data indicated that these specimens from the Mukwadzi River represent a new species to science, herein described as Chiloglaniscarnatus Mutizwa, Bragança & Chakona, sp. nov. This species is readily distinguished from its southern African congeners by the possession of a distinctive extended dermal tissue covering the base of the dorsal fin and the possession of ten mandibular teeth (vs 8, 12, or 14 in the other taxa). Results from this study add to the growing evidence of a high level of undocumented diversity within riffle-dwelling taxa in southern Africa.


Introduction
Rheophilic habitats are characterised by fast flowing water and rocky substratum, which provide a wide range of specialised niches for distinct aquatic taxa adapted to these environments (Thompson 2013;Hrbek et al. 2018).Delimitation of species boundaries in rheophilic taxa using only morphological traits has previously presented challenges due to their superficially similar morphology, which is shaped by exposure to similar environmental drivers (Seegers 2008).However, integrative taxonomy as well as recent collections in under-sampled areas within the African continent have changed the previous perception that rheophilic habitats were depauperate (Schmidt et al. 2015(Schmidt et al. , 2016(Schmidt et al. , 2017(Schmidt et al. , 2023;;Thomson et al. ZooKeys 1197: 57-91 (2024), DOI: 10.3897/zookeys.1197.114679 Tadiwa I. Mutizwa et al.: New suckermouth catfish (Chiloglanis) from the middle Zambezi River 2015; Schmidt and Barrientos 2019; Kashindye et al. 2021;Mazungula and Chakona 2021;Day et al. 2023).These studies, which implemented integrative taxonomic approaches, have allowed the discovery of hidden diversity, particularly within the catfish genera Chiloglanis Peters, 1868 and Amphilius Günther, 1864, from different regions of the continent.An emerging pattern shows that species that were previously perceived to have broad geographic ranges represent species complexes comprising distinct lineages confined to specific river basins (Chakona et al. 2018;Mutizwa et al. 2021).Recently, a careful examination of the once broadly distributed catfish species, C. occidentalis Pellegrin, 1933 andC. micropogon Poll, 1952 from West Africa and A. natalensis Boulenger, 1917 from southern Africa, resulted in the description of 15 new species (Schmidt et al. 2017(Schmidt et al. , 2023;;Mazungula and Chakona 2021).Following these findings, rheophilic habitats have been identified as a new frontier for the discovery of hidden diversity of freshwater fishes in southern Africa and other poorly explored regions on the continent (Morris et al. 2016;Schmidt et al. 2016;Chakona et al. 2018).
Uncertainties about the identity of the broadly distributed C. neumanni in southern Africa have persisted for decades.This species was described from the Bubu River, a tributary of the Great Ruaha River basin in Tanzania, and was considered to be distributed across several eastern, central, and southern African river systems (Daget et al. 1986;Bell-Cross and Minshull 1988).However, following extensive surveys of river systems in east Africa and comprehensive examination of specimens from this region, Seegers (1996) did not record C. neumanni from localities outside the Great Ruaha River system, indicating that this species was not as widely distributed as previously thought.Although the name C. neumanni has persisted in subsequent literature from southern Africa, ichthyologists have consistently made remarks that the suckermouth catfishes of this region required detailed taxonomic investigation to determine their identity (Marshall 2011).In recent years, there has been general consensus among southern African ichthyologists that the species currently referred to as C. neumanni in this region actually represents an undescribed species or even a species complex, including several undescribed species.This assertion is based on the extensive geographic distance between southern Africa and the Bubu River, as well as the emerging patterns of undescribed diversity within other species with similar distribution ranges as C. neumanni.For example, studies of A. uranoscopus (Pfeffer, 1889) and Zaireichthys rotundiceps (Hilgendorf, 1905) led to the resurrection of two synonyms and the description of nine new species (Thomson and Page 2010; Eccles et al. 2011).Indeed, a genetic study by Chakona et al. (2018) identified six unique lineages within the genus Chiloglanis from the Eastern Zimbabwe Highlands ecoregion, a result that is consistent with Marshall's (2011) postulation that the continued use of the name C. neumanni in southern Africa potentially obscures the actual diversity of this group of fishes in this region.A total of four new species to science are currently being described from the Eastern Zimbabwe Highlands ecoregion, with two of them being endemic to this region (Chakona et al., pers. obs.).
During surveys of the southern tributaries of the middle Zambezi River system in 2016 and 2019, morphologically distinct suckermouth catfishes were collected from the Mukwadzi River that drains the western margin of the Great Dyke in Zimbabwe.These specimens could not be attributed to any of the currently described species or recently identified lineages of Chiloglanis from this region.The present work represents the first in a series of studies that aim to resolve the taxonomy of suckermouth catfishes of southern Africa.This study applied integrative taxonomic approaches combining genetic and morphological data to determine the taxonomic distinctiveness of the recently collected specimens from the middle Zambezi River.The significance and implications of incomplete documentation of the diversity of rheophilic species in a region where their unique habitats are under threat from multiple environmental impacts are discussed.

Collections
Specimens were collected from two sites in the Mukwadzi River, a tributary of the Manyame River, a south bank affluent of the middle Zambezi River, during surveys in 2016 and 2019.Samples were collected using a battery-powered Samus 725GN backpack electric fisher with a block net placed downstream to capture dislodged animals in the fast-flowing current.The specimens were photographed to document the live colour pattern then euthanized with clove oil.Muscle tissue from the right side of the specimens was cut out and preserved in 99% ethanol for molecular analysis.Voucher specimens for morphological studies were fixed in 10% formalin in the field and subsequently transferred to 70% ethanol for long term preservation.Additional tissue samples and voucher specimens used in the present study were obtained from the National Fish Collection at the National Research Foundation-South African Institute for Aquatic Biodiversity (NRF-SAIAB) in Makhanda (Tables 1, 2).
Table 1.List of 80 COI sequences used in the present study including six new sequences of the specimens from the Mukwadzi River.The new sequences (in bold) include the hologenetype identified by an asterisk (*) and paragenetypes identified by a plus (+).

DNA extraction, amplification, and sequencing
A total of six new COI sequences of Chiloglanis carnatus sp.nov.were generated for this study.Preparation and sequencing of genetic material was done in the Aquatic Genomics Research Platform at the NRF-SAIAB.Genomic DNA was extracted from preserved tissues using the salting-out method (Sunnucks and Hales 1996).The mitochondrial DNA cytochrome c oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR) using the universal fish DNA barcoding primer set FishF1 and FishR1 (Ward et al. 2005).PCRs were performed with a Veriti 96 well thermal cycler (Applied Biosystems, USA) and each reaction mixture (25 µL) contained 50-100 ng) of template DNA, 6.5 µL of water, 0.5 µL of each primer (10 µM), and 12.5 µL Taq DNA polymerase 2× master mix red (Amplicon PCR enzymes and reagents, Denmark).The PCR amplification profile had an initial denaturation step of 3 min at 94 °C followed by 38 cycles of 30 sec at 94 °C, annealing at 55 °C for 30 sec, and extension at 72 °C for 50 sec, and final extension at 72 °C for 7 min.The amplicons were purified using an Exonuclease I-Shrimp Alkaline Phosphate (Exo/SAP, Thermo Fisher Scientific, USA) protocol (Werle et al. 1994), sequenced using standard fluorescent BigDye v. 3.1 (Applied Biosystems, USA) terminator chemistry in the forward direction, and analysed on a 3500 Genetic Analyser (Applied Biosystems, USA) at the NRF-SAIAB.Additional sequences were obtained from the public databases GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and Barcode of Life Data Systems (BOLD) (http://www.boldsystems.org/)(Table 1).1).Genetic material for C. neumanni from its type locality and C. emarginatus could not be accessed before finalising this study.Mitochondrial DNA sequences were edited, aligned, and trimmed in MEGA-X (Kumar et al. 2016).

Phylogenetic analyses
The sequences were translated into amino acid sequences in MEGA-X to check for stop codons and gaps to ensure that they were copies of functional mitochondrial protein coding sequences.Haplotype groups were identified using DNASP 6 (Rozas et al. 2017).The most suitable model for nucleotide substitution was selected using the Akaike Information Criterion (AIC) (Akaike 1974) as implemented in the program jModelTest 0.1.1 (Darriba et al. 2012).Bayesian phylogenetic inference was performed in MrBayes 3.2.6 (Ronquist et al. 2012) using the TIM3+I+G evolutionary model identified using jModeltest.The phylogenetic tree and posterior probabilities were inferred using four Markov chain Monte Carlo (MCMC) chains which were run for 2 × 10 6 generations with tree sampling every 1000 generations.The program Tracer 1.7 (Rambaut et al.

2018
) was used to analyse the quality of the trace files generated by MrBayes and to determine the number of trees to be discarded as burn-in.The first 25% of the sampled trees for each analysis was discarded as burn-in, and the remaining trees were used to calculate a majority rule consensus tree.Maximum likelihood (ML) analysis of the same dataset was performed in RAxML v. 8 (Stamatakis 2014) through the graphical user interface raxmlGUI v. 2 (Silvestro and Michalak 2012).A total of 10 ML searches were performed in raxmlGUI and support values for the ML tree nodes were estimated by 1000 non-parametric bootstrap inferences (Felsenstein 1985).Bootstrap values equal to or higher that 70% (Hillis and Bull 1993), and posterior probability values at 0.95 or higher (Alfaro and Holder 2006), were considered strong support.

Molecular species delimitation
Four molecular species delimitation methods were used to delineate candidate species within the suckermouth catfishes of southern Africa using the same dataset used for the phylogenetic analysis.The first two methods, Automatic Barcode Gap Discovery (ABGD; Puillandre et al. 2012) and Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. 2021) infer the barcode gap from the data to partition sequences into proposed candidate species.These methods were performed on their respective webservers (https://bioinfo.mnhn.fr/abi/public/abgd/and https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html).The intraspecific diversity priors were set at P min = 0.001 and P max = 0.1) for both methods.
The Kimura (K80) TS/TV distance model was used and the remaining settings were left at their default parameters.The second pair of species delimitation methods included the Bayesian implementation of the Poisson Tree Processes (bPTP) (Zhang et al. 2013) and the General Mixed Yule Coalescent (GMYC) (Pons et al. 2006;Fujisawa and Barraclough 2013).Both GMYC and bPTP require a phylogenetic tree as input and from this tree they estimate rates of branching events to infer which parts of the tree are likely to follow a speciation model (interspecific variation) and which parts follow a coalescent model (intraspecific variation).The bPTP was performed on the web server (http://species.h-its.org/ptp/) using the same tree generated for phylogenetic reconstruction and a MCMC run for 1 × 10 6 generations with 10% burn-in.For the GMYC analysis a fully resolved ultrametric tree was inferred in Bayesian evolutionary analysis by sampling trees (BEAST) 2.4.6 (Bouckaert et al. 2014) using a strict clock and Yule model and the MCMC was ran for 1 × 10 7 generations with tree sampling every 1000 generations.The program Tracer 1.7.2 was used to analyse the quality of the trace files generated by BEAST.TreeAnnotator (Helfrich et al. 2018) was used to summarise the trees sampled by BEAST into a single maximum credibility tree with a burn-in of 25%.The species' limits by threshold Statistics (splits) package (http://r-forge.r-project.org/projects/splits) in R 3.5.0(R Core Team 2018) was used to identify the candidate species from the maximum credibility tree produced by TreeAnnotator.Model corrected intraspecific and interspecific genetic distances of the molecular taxonomic units identified by the species delimitation methods were calculated in PAUP* 4.0a163 (Swofford 2003).2).Because type material for C. fasciatus could not be accessed before finalising this study, 10 conspecific specimens collected from close to the type locality of this species and identified using the species description by Pellegrin (1936) and the key in Skelton (2001) were used as topotypes.The syntypes of C. pretoriae were severely deformed, thus only their meristic counts were included for comparison in this study, but 16 specimens collected from near the type locality of C. pretoriae and identified using the key in Skelton (2001) were used as topotypes for this species.
External meristic counts were performed under a stereo microscope.Vertebrae counts were made from radiographs taken at the NRF-SAIAB using an Inspex 20i Digital X-ray Imaging System (Kodex Inc., New Jersey, USA).Radiographs for the lectotype and paralectotypes of C. neumanni were taken at the Royal Museum for Central Africa in Tervuren, Belgium (MRAC) using a VisiX-MedexLoncin (www.medex.be).A total of nine meristic characters were examined: number of mandibular teeth, pre-maxillary teeth, pectoral-fin rays, pelvic-fin rays, dorsal-fin rays, anal-fin rays, abdominal vertebrae, caudal vertebrae, and total vertebrae (Fig. 3).Following Roberts (1989), vertebrae counts excluded the Weberian structures and began from the fifth vertebra which was identified by a pair of large but slender ribs, and included the hypural complex which was counted as one vertebra.
The abdominal vertebrae were defined as the vertebrae that occurred anterior to the first anal fin ray pterygiophore.Caudal vertebrae were defined as those that occurred posterior to the first anal fin ray pterygiophore and included the hypural complex which was counted as one vertebra (Roberts 1989) (Fig. 3).The genital papillae were examined to determine the sex of the specimens following Friel and Vigliotta (2008).Morphological measurements were standardised by transforming body measurements into percentages of the standard length (SL) and head measurements into percentages of the head length (HL).Principal component analyses (PCA) were performed in PAST v. 3.12 (Hammer et al. 2001) using the covariance matrix for the morphometric data in order to identify morphological characters that contributed the most to distinguishing Chiloglanis carnatus sp.nov.from the other Chiloglanis species from southern Africa.

Phylogenetic analyses
The COI alignment of 80 sequences had 534 base pairs and 176 variable sites.
A total of 47 unique haplotypes were identified.Although the Bayesian phylogenetic tree was not fully resolved, it showed genetic structuring that supported the monophyly of suckermouth catfishes from southern Africa (Fig. 4).ed (posterior probability > 0.95).Genetic divergences within valid and candidate species ranged from 0-1.5% and interspecific divergences ranged from 1.3-15.7%(Table 4).Chiloglanis paratus from the Phongolo River was recovered as the most basal clade that is sister species to all the southern African suckermouth catfishes included in the present study.The relationships among the remaining taxa were not well resolved as they were recovered within five polytomous clades with weak support between them (Fig. 4).The phylogenetic tree inferred using the ML approach had similar topology to the Bayesian inference tree (Fig. 5).inferred similar molecular taxonomic units with the exception of Chiloglanis sp.'dwarf' which was split into two molecular taxonomic units by the latter method.
The integrated approach used in this study provided genetic and morphological characters that clearly and consistently distinguish Chiloglanis carnatus sp.nov.from the known species and lineages from this region.This study has thus provided evidence that supports the description of the Chiloglanis carnatus sp.nov.as a new species.

Taxonomic account
Chiloglanis carnatus Mutizwa, Bragança & Chakona, sp.nov.https://zoobank.org/E1F0912C-986F-450F-9B90-400D86F5F3BCMaterial examined.Holotype.Zimbabwe • ♂, stored in 70% ethanol, 46.8 mm SL, Fig. 9A-E   Chiloglanis carnatus possesses a deeply forked caudal fin that readily separates it from C. pretoriae and C. emarginatus that have emarginate caudal fins, and from C. anoterus that has a caudal fin with extended median rays in males and emarginate in females.Chiloglanis carnatus possesses a caudal fin with an upper lobe that is shorter than the lower lobe.This distinguishes it from C. bifurcus that has a caudal fin with an upper lobe that is longer than the lower lobe.Chiloglanis carnatus has an oral disc with a well-developed mid-ventral cleft that distinguishes it from C. swierstrai that possesses an oral disc without a mid-ventral cleft.Chiloglanis carnatus possesses a smooth skin with a few tubercles occasionally found on the head that separates it from C. fasciatus that has its entire dorsal and lateral body surfaces mostly covered by small tubercles.Chiloglanis carnatus has a dorsal spine with crenate anterior and posterior margins that distinguish it from C. paratus that has a dorsal spine with a serrated posterior margin.
Description.Morphometric proportions and meristics are summarised in Table 7. Holotype meristic counts are given in parentheses.
Body shape.Anterior portion of body slightly compressed dorsally, becoming laterally compressed from pelvic fin insertion to caudal peduncle.Body greatest depth at dorsal-fin insertion.Pre-dorsal profile convex, sharply slopping from snout to posterior nostril, gently from nostril to dorsal-fin origin.Post-dorsal profile about straight from dorsal-fin base to adipose-fin origin, becoming gently concave from adipose-fin origin to caudal fin.Ventral profile gently convex from region just posterior to oral disc to anal-fin origin, becoming gently concave from anal-fin origin to caudal fin.
Head. slightly depressed dorsally.Oval eye dorsally positioned, ~ 1/2 distance between snout and gill opening.Interorbital distance greater than distance between nostrils.Anterior and posterior nostrils closer to the eye than snout.Distance between anterior nostrils slightly greater than distance between posterior nostrils.Posterior nostril medially positioned relative to orbit.Anterior nostril with posterior flap; posterior nostril with anterior flap.Occipital-nuchal shield not visible through skin.Gill opening above pectoral fin insertion.
Oral disc.Mouth inferior; large upper and lower lips combined to form oral disc (see Fig. 9E, K).Oral disc width greater than length.Upper and lower lips with  Dentation.Pre-maxillary teeth arranged in three or four rows; variable number of teeth .Up to 5+5 closely packed mandibular teeth; central teeth projecting higher than outer teeth forming a gentle arc; replacement tooth row emerges anteriorly to the functional row.
Fins.Dorsal-fin ray count 5-7 (6), originating in anterior 1/3 of body, posterior to pectoral-fin origin.Dorsal fin basal portion covered by a fleshy skin prominent in large adult males and females with ~ ¾ of the dorsal spine and the first two rays also covered by fleshy skin (Fig. 10).Dorsal spine length ~ 80% of longest dorsal fin ray length.Dorsal spine with dentate anterior and posterior margins.Pectoral-fin ray count 6-8 (8), origin anterior to gill opening; pectoral spine anterior margin smooth; dentate posterior margin; pectoral spine length ~ 80% of pectoral fin length.Adipose fin origin preceded by anterior tissue flange; rounded (Fig. 10).Caudal fin forked, lower lobe longer than upper lobe.Anal-fin ray count 12 or 13 (13), origin posterior to origin of adipose fin; terminating just before end of adipose-fin; rounded.Pelvic-fin ray count 6 or 7 (7), origin posterior to midpoint between end of dorsal-fin and adipose-fin origin; rounded.
Skin.Skin smooth with occasional tubercles present, concentrated on dorsal and lateral surface of head.Lateral line complete; originating anterior to dorsal fin at same horizontal level of orbit and sloping ventrally until it lies mid-laterally along body.Sexual dimorphism.Urogenital opening situated adjacent to origin of anal fin.Urogenital papillae sexually dimorphic; elongated in males; reduced and separated from anus by shallow invagination in females.
Colouration.Overall body background colouration brown with yellowish ventral surface.Anterior portion of body dark brown becoming paler towards posterior.Small dark melanophores scattered across entire dorsal and lateral sides.Six yellowish brown blotches on lateral surface of body; two vertically arranged posterior to end of adipose fin; one above origin of anal fin; two above pelvic fin origin; and one below dorsal fin origin.Basal 1/3 of fins pale to dark brown with medium and posterior portion of fins gradually becoming translucent.Dark blotch cuts vertically across caudal peduncle lobes.
Etymology.The specific epithet carnatus means fleshy, referring to the dermal tissue covering the base of the dorsal fin of some of the larger specimens of this species and the general robust body structure of this species compared to its regional congeners.
Distribution.Chiloglanis carnatus was collected from two sites in the Mukwadzi River near the bridge on the Mutorashanga Road.The Mukwadzi River is a perennial river that originates from wetlands (dambos) on the eastern side of the Great Dyke.This river flows in a north-western direction cutting through the Great Dyke before it joins the Manyame River.The Great Dyke is a major intrusion of mafic and ultramafic rocks that have vast ore deposits, including gold, silver, chromium, platinum, nickel, and asbestos.The rich mineral deposits have resulted in the establishment of many mines along the Great Dyke.The sites where C. carnatus was collected were in a communal area surrounded by rural communities on the western slope of the Great Dyke.The substratum at the sites was composed of bedrock, cobbles and gravel, and the riparian vegetation was dominated by Syzygium Gaertner, 1788 and Phragmites Adanson, 1763.At these sites C. carnatus co-occurred with native fish species that include Labeo cylindricus Peters, 1852, Opsaridium zambezense (Peters, 1852), Enteromius trimaculatus (Peters, 1852), Tilapia sparrmanii Smith, 1840, Clarias gariepinus (Burchell, 1822), and Labeobarbus marequensis (Smith, 1841) as well as the non-native species Serranochromis jallae (Boulenger, 1896) and Micropterus salmoides (Lacepède, 1802).

Discussion
This study integrated molecular and morphological data to evaluate the taxonomic distinctiveness of specimens of suckermouth catfishes that were collected from the middle Zambezi River system in Zimbabwe.Based on substantial genetic differentiation as well as consistent meristic, morphometric, and qualitative differences from its southern African congers, a new species of Chiloglanis is described.This is the first description more than five decades after the last comprehensive review of Chiloglanis species from southern Africa (see Jubb and Le Roux 1969).This study adds to the growing body of literature that demonstrates the value of integrative taxonomic approaches in the discovery and description of new species within this region (Maake et al. 2014;Morris et al. 2016;Riddin et al. 2016;Kambikambi et al. 2021;Mazungula and Chakona 2021).As evidenced from this study and work by Chakona et al. (2018), additional species of suckermouth catfishes from southern Africa remain to be formally described.It is anticipated that ongoing taxonomic studies on this group of fishes will result in the description of at least ten new species from this region.These species were all previously included under a single species, C. neumanni, but this study and ongoing work by researchers from the NRF-SAIAB indicates that this species does not occur in southern Africa.Updated taxonomic information of Chiloglanis species from this region will improve our understanding of biogeographic and phylogeographic patterns as well as drainage evolution in the region.
The dentition of species in the genus Chiloglanis, like that of most members of the family Mochokidae, is highly specialised (Roberts 1989).Chiloglanis carnatus possesses ten closely packed mandibular teeth, a number not found in any other Chiloglanis species in southern Africa.Variation in the number of mandibular teeth in individual specimens can be observed due to tooth loss from the functional row, delayed exposure of some teeth in the replacement row, or early advancement of some replacement row teeth (Roberts 1989).However, by examining both the functional and replacement rows, it was possible to determine the diagnostic number of teeth for this species.Outside southern Africa, the presence of ten mandibular teeth has been reported in west African species such as C. kolente Schmidt et al., 2017, C. kabaensis Schmidt et al., 2017, C. nzerekore Schmidt et al., 2017, C. occidentalis Pellegrin, 1933, and C. normani Pellegrin, 1933(Paugy et al. 2003;Schmidt et al. 2017).In addition to dentation, there were several morphometric characters associated with the oral disc (e.g., maxillary barbel length, oral disc width, lower lip length and mandibular tooth row width) that distinguish C. carnatus from congenerics in southern Africa.Considering the importance of the oral disc in the ecology of the species in this genus, these differences warrant further study, particularly assessing potential differences in trophic ecology.
Rheophilic habitats form 'islands' with suitable environmental conditions for specialised taxa such as those in the genus Chiloglanis.The disjunct distribution of these habitats within a river may play an important role in promoting genetic and morphological diversity within rheophilic taxa.Some rheophilic species have very narrow distribution ranges such that significant differences have been found in the fish communities occurring at different rapids within the same river system (Hrbek et al. 2018).In southern Africa high genetic and morphological diversity within C. anoterus has been reported from geographically isolated populations in the upper sections of the Phongolo and Inkomati river systems, highlighting the importance of the rheophilic habitats in headwater streams (Morris et al. 2016).The close association of Chiloglanis species with rheophilic habitats probably promotes diversification; however, this has yet to be explicitly tested within this region.The discovery of the C. carnatus from a small section of the Mukwadzi River as well as other undescribed species within southern Africa (Chakona et al. 2018) emphasises the need for accelerating inventory of the diversity found in rheophilic habitats as these may harbour a considerable number of species which are still unknown to science.
A number of southern African freshwater fish species in the genera Enteromius Cope, 1867, Nothobranchius Peters, 1868, Pseudobarbus Smith, 1841, Sandelia Castelnau, 1861, Galaxias Cuvier, 1816, and Oreochromis Günther, 1889 are threatened with extinction due to their narrow geographic ranges, the introduction of invasive species, and habitat degradation (Marshall and Tweddle 2007;Jordaan and Chakona 2017;Roux and Hoffman 2017b;Nagy and Watters 2019).Among the Chiloglanis species from southern Africa, C. bifurcus and C. emarginatus are under threat with the former classified as Critically Endangered and the latter as Vulnerable in the IUCN Red List of threatened species (Roux andHoffman 2017a, 2018).Chiloglanis bifurcus is a narrow-range endemic species that is confined to the upper sections of the Inkomati River system, whereas C. emarginatus' range in the Phongolo River system has declined substantially over the past decades (Roux andHoffman 2017a, 2018).Habitat loss though flow regulation, pollution, and sedimentation has been attributed as the main driver of population decline in both these species (Roux andHoffman 2017a, 2018).Chiloglanis carnatus was collected from two sites in the Mukwadzi River.The section downstream of these sites as well as other tributaries of the Mukwadzi River are heavily impacted by anthropogenic activities.There are at least 13 small impoundments in the Mukwadzi River before its confluence with the Manyame River.Largemouth bass (Micropterus salmoides) and the nembwe (Serranochromis jallae) were also introduced into this river system, and this combination of flow modification, water abstraction, and non-native species is likely to negatively impact populations of native spe-cies (Gratwicke and Marshall 2001;Gratwicke et al. 2003;Kadye et al. 2013;Kadye and Booth 2020).In addition to the modification of this river and the non-native species, the rich mineral resources found within the Great Dyke attract formal and informal mining operations which also threaten the species living within these rivers through increased sedimentation/siltation which may cause habitat loss.Although little is known about the distribution of C. carnatus beyond the sites sampled in this study, multiple anthropogenic activities in the Mukwadzi River catchment raise concerns about the conservation status of this species.
The description of C. carnatus contributes towards clarifying the taxonomic uncertainty surrounding species of the genus Chiloglanis found within the geographic range formerly attributed to C. neumanni within southern Africa.The discovery of C. carnatus follows the common pattern found among recent taxonomic studies within the region whereby comprehensive sampling across poorly explored regions and the use of integrated taxonomic approaches has identified unique diversity within species previously thought to have wide distribution ranges (Bragança et al. 2020;Kambikambi et al. 2021;Mazungula and Chakona 2021).This pattern is likely to be consistent across southern Africa suggesting underestimation of the region's biodiversity.In particular, species such as those from the genus Chiloglanis are likely to be more diverse since they occur in disjunct distributions in rheophilic habitats, which are likely to be associated with allopatric speciation.This study also raises the awareness of the potential unique riverine diversity of the rivers that flow through the Great Dyke, an important geological feature where 20 endemic plant species that are adapted to the unique serpentine soils have been recorded (Wild 1965).Further exploration of the aquatic fauna of this poorly surveyed region is likely to uncover additional new species for science.

Figure 1 .
Figure 1.Distribution of Chiloglanis species in southern Africa based on data from the National Research Foundation-South African Institute for Aquatic Biodiversity extracted from the GIBF database (https://www.gbif.org).
The first clade contained Chiloglanis carnatus sp.nov.from the Manyame River and C. fasciatus from the Okavango River.The second clade contained C. anoterus and C. bifurcus from the Incomati River system as well as C. pretoriae from the Limpopo River system.The third clade contained Chiloglanis sp.'Nyangombe' from the Nyangombe River and Chiloglanis sp.'dwarf' from the Pungwe and Ruo rivers.The fourth clade contained Chiloglanis swierstrai from the Limpopo River system.The fifth clade contained the Chiloglanis sp.'Zambezi', Chiloglanis sp.'Pungwe', Chiloglanis sp.'roughskin', and Chiloglanis sp.'Shire' lineages.The Chiloglanis sp.'roughskin' lineage occurs in the Buzi and Pungwe rivers, whereas Chiloglanis sp.'Pungwe' is endemic to the Pungwe River.Chiloglanis sp.'Shire' and Chiloglanis sp.'Zambezi' lineages were found in the lower Zambezi River system with the latter lineage also occurring in the Okavango River.

Figure 3 .
Figure 3. Illustration showing how fin rays and vertebrae were counted using x-ray radiographs.The red dots along the vertebra represent the abdominal vertebrae and the blue dots represent the caudal vertebrae.

Figure 4 .
Figure 4. Bayesian inference tree of the species and lineages of the genus Chiloglanis found in southern African.The numbers at the nodes represent the Bayesian posterior probabilities.The black bars represent candidate species proposed by four molecular species delimitation methods: Automatic Barcode Gap Discovery (ABGD), Automatic Partitioning (ASAP), Bayesian implementation of the Poisson Tree Processes (bPTP), and General Mixed Yule Coalescent (GMYC).

Figure 5 .
Figure 5. Maximum likelihood tree of the species and lineages of the genus Chiloglanis found in southern African.The numbers at the nodes represent the bootstrap values.

Figure 6 .
Figure 6.Scatter plot of the first two principal components of the morphometric characters of Chiloglanis species and lineages from southern African.

Figure 8 .
Figure 8. Scatterplots of the meristic characters of the Chiloglanis species from southern African.

Table 2 .
List of 184 specimens examined in this study including 19 specimens collected from the Mukwadzi River.

Table 3 .
Morphological characters examined in the present study.

Table 4 .
Ranges of cytochrome oxidase I (COI) genetic distances (%) between the Chiloglanis species included in the present study.

Table 5 .
Summary of morphological characters examined in the present study.All values except standard length (SL) and Head length (HL) are given as percentages of the HL or SL.For the meristics the mode is given alongside the range of the counts in parentheses where the counts varied.

Table 6 .
The PCA loadings for the first two principal components for the measured characters of Chiloglanis species and lineages from southern Africa.

Table 7 .
Summary of morphological characters for Chiloglanis carnatus sp.nov.All values except standard length (SL) and Head length (HL) are given as percentages of the HL or SL.