A new species of redfin (Teleostei, Cyprinidae, Pseudobarbus) from the Verlorenvlei River system, South Africa

Abstract Pseudobarbus verloreni, a new species, is described from material collected in the Verlorenvlei River system on the west coast of South Africa. It differs from its congeners (except Pseudobarbus skeltoni, Pseudobarbus burchelli, and Pseudobarbus burgi) by the presence of two pairs of oral barbels. Pseudobarbus verloreni sp. n. can be distinguished from the three currently described double barbeled Pseudobarbus species by the following combination of characters: pigment pattern, generally deeper body relative to standard length, a longer intestine associated with the deeper body form, shorter snout relative to head length, and much shorter anterior barbels relative to head length. The new species is distinguished from Pseudobarbus burgi in the neighbouring Berg River system by its longer head and longer pre-dorsal length. It seems as if Pseudobarbus verloreni sp. n. has been extirpated from the Langvlei River system and face several threats to its survival in the Verlorenvlei River system.


Introduction
Species of the cyprinid genus Pseudobarbus (commonly referred to as "redfins") are distinctly pigmented small-to-medium sized riverine fishes endemic to southern Africa (Skelton 1988). Pseudobarbus was described by Smith (1841) as a subgenus of Barbus.
Langvlei population has also been lost. Skelton (1988) noted that specimens of P. burgi from the Verlorenvlei River system had a longer intestine and longer predorsal length compared to specimens from the Berg River system. Bloomer and Impson (2000) discovered high levels of genetic differentiation (5.3-7.0% for the mitochondrial control region) between populations of P. burgi from the Verlorenvlei and Berg river systems, indicating a long history of isolation. The differentiation between these two lineages was confirmed by Swartz et al. (2009) and . The purpose of the present study is to describe the Verlorenvlei Pseudobarbus population as a new species, Pseudobarbus verloreni sp. n.

Materials and methods
Institutional abbreviations follow Sabaj (2013) and are listed at http://www.asih.org/ node/204. Description of the new Pseudobarbus species is based on 47 specimens (holotype and paratypes) that were collected from the Verlorenvlei River system during surveys conducted in January 1999 and March 2012. The type material has been deposited at the South African Institute for Aquatic Biodiversity (SAIAB), MRAC, USNM and BMNH.

Molecular data
Two Pseudobarbus individuals from the Verlorenvlei River system were sequenced to assign a hologenetype and a paragenetype following Chakrabarty (2010) for the mitochondrial cytochrome b gene. The sequences were added to the genetic analysis done by  to show the phylogenetic position of the hologenetype and the paragenetype in relation to all known lineages and species of Pseudobarbus. Methods of DNA extraction, amplification, sequencing and analysis follow  and . The hologenetype and paragenetype sequences were deposited in GenBank for future reference (GenBank numbers are given below) following the definitions of Chakrabarty (2010).

Morphological data
Meristic and morphological characters were examined following Hubbs and Lagler (1958), Skelton (1988), and . The characters considered for each specimen in the present study (22 morphometric measurements) and (12 meristic counts) are presented in Table 1. In addition, entire branchial baskets were dissected from three specimens to examine and count pharyngeal teeth.
We compared morphological and meristic differences among all double barbeled redfins using raw data from Skelton (1980Skelton ( , 1988 and . Specimens were assigned to four groups based on geographic origin and previous genetic results (Bloomer and Impson 2000;Swartz et al. 2009Swartz et al. , 2014: P. skeltoni (n=25), P. burchelli (n=128), P. burgi (specimens from the Berg River system only; n=66) and Verlorenvlei Pseudobarbus (specimens from the Verlorenvlei River system only; n=47). A total of 47 specimens of Verlorenvlei Pseudobarbus were radiographed to count skeletal features. Statistical analyses were performed with the programs InfoStat (Di Rienzo et al. 2012), PAST and STATISTICA 12. Prior to analyses, morphometric data were normalised using procedures described by Lleonart et al. (2000). Analyses of meristic characters were performed using the raw data.
Principal component analysis (PCA) was performed using the correlation matrix to explore the separation of the specimens based on the normalised morphometric data (Lleonart et al. 2000) and raw meristic characters. Invariant characters (such as the number of pelvic fin rays) were excluded from analysis. All scores (including PC1) were considered, because the normalisation approach allows for size free comparisons (Lleonart et al. 2000).
Discriminant Function Analysis (DFA) was performed to visualise the degree of morphological separation among the species and to identify the most important characters that contribute to the differentiation. DFA also provides jacknifed measurements of re-classification success of individuals to their original group, as well as identifying the group to which individuals were assigned if misclassified. Separate DFAs were performed for the morphometric and meristic characters, as well as for these two data sets combined. Figure 1 is a re-analysis of the phylogeny done by  to include the genetypes of Pseudobarbus verloreni sp. n. sequenced in the present study. It shows the phylogenetic relationships among double barbeled redfins based on the mitochondrial cytochrome b data, and shows the position of the new species that is distinct from the three described double barbeled redfin species, including the three previously identified lineages of P. burchelli (Swartz et al. , 2014. The model corrected genetic distances show deep divergences (6.6-12.3%) between the new species and the other members of the double barbeled redfin group.

Results
Principal components analysis (PCA) of normalised morphometric and raw meristic characters shows Pseudobarbus specimens from the Verlorenvlei River system, herein described as the new species P. verloreni sp. n., form a cluster that is clearly separated from P. skeltoni and marginally overlaps with P. burchelli and P. burgi ( Figure 2). The most important factor loadings are presented in Table 2. PCI was mainly defined by differences in head length, head depth, predorsal length, number of lateral line scales, number of scale rows between lateral line and dorsal fin, number of scale rows around the caudal peduncle and the number of predorsal scale rows  (Table 2). PCII primarily contrasted differences in body depth, length of anterior barbel, and snout length. PCIII was mainly defined by caudal peduncle depth and body width. Specimens of P. verloreni sp. n. were associated positively with PCII, describing individuals characterised by deeper bodies relative to standard length. Specimens of the new species are separated from those of P. burgi, which were associated positively with PCI, describing individuals characterised by deeper heads relative to head length. Note also that the syntypes of P. burgi are clearly not conspecific with specimens of P. verloreni ( Figure 2).
The Discriminant Function Analysis (DFA) performed using combined morphometric and meristic characters correctly classified all individuals of the new species (Table 3). Similar to PCA results, the discriminant scores showed that the new species was clearly separated from the three previously described species of Pseudobarbus with two pairs of oral barbels (results not shown).
The DFA using morphometric measurements revealed morphological shape differences between the new species and the other previously described species of Pseudobarbus with two pairs of oral barbels. This analysis correctly classified all individuals of the new species, P. verloreni sp. n., as well as P. burgi sensu stricto while three individuals of P. skeltoni and four individuals of P. burchelli were misclassified (Table 3). In contrast, the DFA using meristic characters showed poor classification of individuals of the four species, with three individuals of the new species, 29 individuals of P. burchelli and one individual of P. burgi being misclassified (Table 3).
Based on the deep genetic and significant morphological divergence between individuals from the Verlorenvlei River system and other members of the double barbeled redfin group, the Verlorenvlei population represents a new species.
Holotype. South Africa: Western Cape Province: SAIAB186092, mature male, 70.8 mm standard length (SL), collected from the Verlorenvlei River, 20 m upstream from railway at the Het Kruis bridge on R365 (32.60179000 S, 18.75039000 E) on 13 March 2012 by E. Swartz and W. Bronaugh, using a seine net. Hologenetype: GenBank number KM366106.
Description. Proportional measurements and meristic characters are presented in Table 4. The body is fusiform, more or less laterally compressed, with a conspicuous lateral stripe from the posterior edge of the head terminating in a dark spot at the base of the caudal peduncle. The lateral band is more pronounced in juveniles and sub-adults, but is less conspicuous in adults. Distinct linear speckles are present on the abdomen. The head is relatively small and slightly depressed; head length is almost equal to body depth. Two pairs of barbels, rostral (anterior) barbels minute and much smaller than eye diameter; maxillary barbels are rooted at the corner of the mouth are longer than rostral barbels and are equal or smaller than eye diameter. Eyes are relatively large, located dorsolaterally, closer to the tip of the snout than the caudal margin of the operculum, interorbital space is flat. Mouth is sub-terminal, lower lip is unretracted and lacks a cartilaginous plate. Snout is relatively short, only few nuptial tubercles present (observed in only one individual; Figure 4) or tubercles are completely lacking.
Counts for the holotype are given in a separate column in Table 4. Dorsal fin of the new species has 3 unbranched and 7 or 8 branched rays, distal margin almost straight, anterior base of dorsal fin inserted directly above or slightly in front of the origin of pelvic fins. Origin of dorsal fin inserted almost midway between tip of snout and base of caudal fin. Pectoral fins with 13 to 16 rays, shorter than head length, reaching beyond halfway to pelvic fin origin. Pelvic fin with 7 to 9 rays, shorter than head, posterior edge gently rounded, reaching the anus in males and within one or two scale rows to the anus in females. Anal fin with 3 or 4 unbranched and 5 branched rays, distal margin almost straight or gently rounded, origin inserted closer to origin of pelvic fin than base of caudal fin. Caudal fin forked, shorter than head length. Genital opening situated adjacent to anterior base of anal fin.
Scales moderately large; lateral line complete, majority of specimens have 32 scales along lateral line (range 29-36); 5-6 (mode 6) scale rows between dorsal fin origin and lateral line; 4-5 (mode 5) scale rows between pelvic fin origin and lateral line; 4-5 (mode 4) scale rows between lateral line and anal fin origin; 12-16 (mode 12) circumpeduncular scale rows. Predorsal scale rows 13-18 (mode 16), embedded in skin, smaller than flank scales. Patch between head and posterior base of pectoral fins naked; scales between posterior base of pectoral fins and anterior base of pelvic fins smaller than flank scales and embedded. Pelvic fins lack prominent or elongate axillary scales. Scales radiately striated.
Additional information. SAIAB59813, juveniles (n=68, 13.5-28.4 mm SL) and adults (n=3, 59.3-64.6 mm SL), collected from the Verlorenvlei River, near Grootfontein farm (32.39830017 S, 18.47419930 E) on 23 January 1999 by R. Bills and D. Naran using a seine net and D-net. Juveniles and sub-adults of P. verloreni have a conspicuous lateral band, while the lateral band is either less prominent or interrupted by linear spots in juveniles and sub-adults of the other double barbeled Pseudobarbus species. The new species has three rows of pharyngeal teeth, teeth pattern 2.3.5-5.3.2 (observed in 3 adults; SAIAB59813); teeth with asymmetrical crowns and hooked at their tips. Pseudobarbus verloreni sp. n. has the longest intestine relative to standard length compared to all the Pseudobarbus species (Skelton 1988: Figure 25Bc).
Comparisons. Pseudobarbus verloreni sp. n. is distinguished from all other species of Pseudobarbus (except P. skeltoni, P. burchelli and P. burgi) by the presence of two pairs of oral barbels. The new species is distinguished from P. skeltoni, P. burchelli and P. burgi by having a deeper body relative to standard length, smaller anterior barbels and shorter snout relative to head length ( Table 4). The new species is distinguished from P. skeltoni by having a sub-terminal mouth (versus terminal in adults of the latter species) and a smaller head relative to standard length (Table 4). Pseudobarbus verloreni is distinguished from P. burchelli and P. skeltoni by a deeper head, wider distance between the eyes (inter-orbit), larger eye relative to head length, shorter posterior barbel relative to head length, wider post-orbit distance, shallower caudal peduncle and generally fewer scales along the lateral line. Pseudobarbus verloreni is distinguished from P. burchelli and P. burgi by lack of cartilaginous plate on lower lip and having unretracted lips. The new species is distinguished from P. burgi by its longer head, longer predorsal length, shorter caudal peduncle and larger eye (Table 4).
Reproduction. Unknown, but spawning possibly occurs around October-December, based on the general patterns of congeners.
Distribution and habitat. Pseudobarbus verloreni is a lowland species that is restricted to the Verlorenvlei River system on the west coast of South Africa ( Figure 5). The morphological features of two juvenile specimens of Pseudobarbus collected from the Langvlei River by Thorne and Cambray in 1986 (SAIAB 130464) are consistent with juveniles of the new species, and are thus assigned to P. verloreni. The Langvlei River population is likely to have been extirpated, as no specimens of Pseudobarbus have been collected during more recent surveys (2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012). The major impact on this river is excessive water extraction that causes the river to dry up completely during the dry season. The Verlorenvlei River system has a gentle gradient and slow to moderate flow for much of the year. The water is highly turbid during the rain season (winter months) when water volume and flow velocity is high, but it becomes less turbid during low flow periods. Most sections of the river system recede into a series of isolated pools during the dry season, especially during late summer and autumn. The bottom substratum is predominantly sand, silt and mud. This is in contrast with the majority of the streams in the CFR that are associated with the Cape Fold Mountains with steeper gradients, clear water, moderate to fast flow throughout the year and rocky substratum. The species was possibly widespread throughout the Verlorenvlei and Langvlei River systems in the past, but numbers likely declined during the last century due to predation and competition from introduced fish species and habitat degradation (see below).
Etymology. The species is named after the Verlorenvlei River system to which it is now confined.
Conservation. The Verlorenvlei redfin was listed as Endangered during the most recent IUCN assessment by Tweddle et al. (2009). The presence of non-native predatory black bass Micropterus spp and potential competitors, banded tilapia Tilapia sparrmanii, Mozambique tilapia Oreochromis mossambicus and common carp Cyprinus carpio, habitat degradation and excessive water withdrawal for agricultural purposes pose the greatest threat to the survival of this species. Protection of critical habitats and establishment of sanctuaries are some of the most immediate conservation measures required to prevent further decline. The effectiveness of current protected areas in conserving Pseudobarbus verloreni is limited because they largely encompass upland areas where this species does not occur. Long-term measures to protect and prevent extinction of this species may have to include eradication of alien fishes and the construction of barriers to prevent re-invasion where feasible and restoration of existing habitats to facilitate recovery.  128824, 186092 and 192542;site 4=SAIAB 130453 and 59808). Also shown is site 5 where the species was collected in the Langvlei River system in 1986 (red circle; site 5=SAIAB 130464), but have not been found in subsequent surveys. The insert map shows the study area in relation to Cape Town, Cape Agulhas (most southern point in Africa) and neighbouring major river systems.

Discussion
Specimens of Pseudobarbus from the Verlorenvlei River system show clear genetic and morphological differences when compared with the three currently described double barbeled Pseudobarbus species (P. burchelli, P. burgi, and P. skeltoni) and are thus described as a new species. The morphological differentiation between P. verloreni sp. n. and P. burgi reported here is consistent with the findings of Skelton (1988) who reported considerable 'intraspecific' morphological variation between Verlorenvlei and Berg populations. The most informative characters that distinguish P. verloreni sp. n. from P. burgi are body depth, head length, predorsal length, snout length and anterior barbel length. However, P. verloreni sp. n. and P. burgi cannot be distinguished based on meristic characters because considerable overlap exists between the two species.
Phylogenetic results from the present study, Swartz et al. (2009) and  show that the relationships among P. verloreni sp. n. (referred to as Verlorenvlei lineage in latter two studies), P. burgi sensu stricto, P. burchelli sensu lato and P. skeltoni are not well resolved, with a polytomy linking the new species and the other taxa. This is further evidence that the Pseudobarbus from Verlorenvlei represents a separate species as it does not clearly group with one of the other species or lineages. Our review of available material of double barbeled redfins confirmed that P. verloreni sp. n. is restricted to the Verlorenvlei River system and likely have been extirpated from the adjacent Langvlei River system.
Reduced tubercle occurrence in P. verloreni could represent a different breeding strategy or behaviour compared to other redfins. Further research is required to better describe the ecology, biology, population size, distribution and conservation status of this species. There are serious conservation concerns, because this species is associated with pool habitats, which are also favourable habitats for non-native fish predators and competitors. This species is also threatened by proposed mining activities and excessive water withdrawal in the Verlorenvlei catchment. Improved understanding of the conservation status, distribution and ecology is a critical requirement for developing effective conservation measures to prevent extinction of this species. The current protected areas are unlikely to be effective for the conservation of Pseudobarbus verloreni as the known distribution range of this species falls outside protected areas. Expansion of protected areas and education of landowners may be necessary to ensure survival of this species.