﻿Taxonomic revision of the Southeast Asian brook barb genus Poropuntius Smith, 1931 (Teleostei, Cyprinidae) with description of a new species from Vietnam

﻿Abstract Molecular data from samples encompassing 22 nominal species of Poropuntius indicate that the species-level diversity in the genus has been vastly overestimated, likely due to inadequate taxon and geographic sampling and reliance on morphological characters that vary intra-specifically. The latter includes discrete mouth morphologies related to alternate feeding strategies (ecomorphs) within populations. One new species is described, Poropuntiusanlaoensis Hoàng, Phạm & Trần, sp. nov., and 17 synonyms of six valid species names of Poropuntius, P.krempfi, P.alloiopleurus, P.huangchuchieni, P.laoensis, P.kontumensis, and P.deauratus, are recognised. Additional taxonomic changes in this widespread and generally poorly known genus are likely as more molecular and morphological data become available.


Introduction
Thirty-three names currently are recognised as valid for species of Poropuntius (Fricke et al. 2023) with distribution of the genus ranging from the Irrawaddy River basin in Myanmar to the Mekong and Red River basins in Yunnan, China and south through Vietnam, Laos, Cambodia, Thailand, and peninsular Malaysia to Sumatra, Indonesia.The greatest diversity occurs in the Mekong basin of Yunnan and Vietnam, and only one species is known from peninsular Malaysia and one from Sumatra (Fricke et al. 2023).
Poropuntius has been characterised as usually having 8½ branched dorsal rays, large serrae on the posterior edge of the last simple dorsal ray, and tubercles covering the tip of the snout and occurring on the lacrimal bones (Rainboth 1996;ZooKeys 1204ZooKeys : 199-222 (2024)), DOI: 10.3897/zookeys.1204.120873 Huy Duc Hoang et al.: Taxonomic revision genus Poropuntius (Cyprinidae) a new species Roberts 1998).Tubercles occur on both sexes and, at least in most species, on juveniles as well as adults (Roberts 1998).In addition, most species have rostral and maxillary barbels, a black to dusky submarginal stripe on the upper and lower caudal-fin lobes, and a well-developed keratinised edge on the lower jaw.
Considerable confusion surrounds the taxonomy of Poropuntius, with most species having been diagnosed using morphological characters that tend to be highly plastic and comparisons made to few congenerics.Wu et al. (2013) and Muhammad-Rasul et al. (2018) investigated molecular diversity of Poropuntius and, although they targeted only a few species and small geographic areas, their results suggested some morphological hypotheses concerning species delimitations are incorrect, and larger molecular studies are needed to improve our understanding of species diversity in Poropuntius.Recent fieldwork throughout much of the distribution of Poropuntius, especially in species-rich Vietnam, has provided material for a broader study of molecular variation in the genus.Results of that study, reported herein, suggest that several species names, including nominal species of Acrossocheilus and Hypsibarbus in Vietnam, are synonyms of previously described species.A newly discovered species from Vietnam is described using morphological and molecular characteristics.

Field sampling
Tissues were taken from selected specimens collected in the field or purchased in local markets throughout much of the range of Poropuntius (Fig. 1) and stored in 95% ethanol for molecular analysis.Specimens were then fixed in 10% formalin, subsequently transferred to 70% ethanol, and deposited at the University of Science, Ho Chi Minh City, Vietnam (UNS), Florida Museum of Natural History, Florida, USA (UF), and Kunming Institute of Zoology, Yunnan, China (KIZ).Samples newly obtained for this study included those from the same drainages as type localities for all nominal species in the molecular analysis except P. krempfi (Pellegrin & Chevey, 1934) for which samples were from near the type locality in the Red River drainage (Suppl.material 1: table S1).Multiple samples and localities were included, when possible, for widespread species.Sampling sites were assigned to freshwater ecoregions following Abell et al. (2008) and Hoang et al. (2021aHoang et al. ( , 2021b)).

Specimens of Acrossocheilus baolacensis
Acrossocheilus macrophthalmus Nguyễn & Ngô, 2001 was described from Vietnam, Hòa Bình, Song Hong ecoregion, Black River.Near-topotypic material (UNS 2018-1310) was collected in the Gâm River of the Song Hong ecoregion and identified through comparison with the description in Nguyễn and Ngô (2001).
Lissochilus aluoiensis Nguyen, 1997 (recently as Poropuntius aluoiensis) was described from Vietnam, Thua Thien Hue Province, A Luoi, A Sap at Nham, Se Kong basin.Topotypic material (UNS 2018-0304) was collected from the A Sap River of the upper Sekong drainage and identified through comparison with the description in Nguyen (1997: 1-4).
Poropuntius krempfi (Pellegrin & Chevey, 1934) was collected in the Gâm River of the Song Hong ecoregion.Identification of our specimens (UNS 2018-1410) was based on comparison with material from the Red River of Song Hong, northern Vietnam, the type locality.

DNA extraction and amplification
DNA was extracted from fin clips stored in 95-99% ethanol using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA) and following the protocol suggested by the manufacturer.Two mitochondrial genes, cytochrome oxidase subunit I (COI) and cytochrome b (Cytb), were amplified using polymerase chain reaction (PCR).Primers and PCR conditions followed Ward et al. (2005) for COI and Durand et al. (2012) for Cytb.PCR products were visualised on 1-2% agarose gels, and the most intense products were selected for purifying and Sanger sequencing by 1ST BASE (https://base-asia.com/).

Phylogenetic analyses
Chromas 2.6.6 (http://technelysium.com.au/) was used to inspect the sequence chromatograms and assemble them into contigs, and MUSCLE in MEGA 7 (Edgar 2004;Kumar et al. 2016) was used to align the consensus sequences for each gene.Alignments were inspected by eye for accuracy, and sequences were trimmed at the 3' and 5' ends to minimise missing characters.The final data matrix consisted of 568 bp for COI and 872 bp for Cytb used in the separated analyses.Uncorrected pairwise sequence divergence was estimated using the substitution model of Kimura 2-parameters, bootstraps 1000 implemented in MEGA 7 (Kumar et al. 2016).All sequences generated for this study were deposited in GenBank (Suppl.material 1: table S1).For each independent dataset of COI and Cytb, phylogenetic inferences based on Maximum Likelihood (ML) were made using IQ-TREE (Nguyen et al. 2015) through the IQ-TREE web server (Trifinopoulos et al. 2016, http://www.iqtree.org/).Optimal partitioning models for the ML inference were selected by ModelFinder (Chernomor et al. 2016;Kalyaanamoorthy et al. 2017) in IQ-TREE, using the minimum BIC score.Partition analysis suggested best fit models for ML inference: TN+F+I+G4 (BIC = 6531.319,lnL = -2650.474)for COI and HKY+F+I+G4 (BIC = 6910.503,lnL = -3065.931)for Cytb.Ultrafast bootstrap (BS) analysis for 1000 iterations (Bui et al. 2013) was carried out to determine statistical support for the nodes in ML.The trees obtained from ML were visualised using Figtree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree).

Results
The ML trees for the COI and Cytb sequences are shown in Fig. 2. The COI topology is consistent with recognition of 16 species (Fig. 2A), all represented by more than one sequence with ≥ 83% bootstrap support, including one undescribed species.
The Cytb sequences were used to examine the results of the study of P. huangchuchieni in southwest China by Wu et al. (2013) with a greater Cytb dataset (Fig. 2B).The five major lineages identified by Wu et al. (2013) are reassigned.The LX lineage of Wu et al. (2013) from the Song Hong aligned with P. krempfi in our sequences, the SW of the Upper Salween with P. opisthopterus, the RL of the Song Hong with P. alloiopleurus, the MK-A of the Lower Lancang with P. huangchuchieni, and the MK-B of the Lower Lancang with P. laoensis.The greatest similarity in the Cytb sequences was 98% (±0.00), found between P. laoensis, which occurs in the Lower Lancang, Kratie -Stung Treng, northern Annam, Chao Phraya, and Middle & Lower Salween ecoregions, and P. huangchuchieni in the Lower Lancang (Table 1).The COI data for P. huangchuchieni (Fig. 2A) aligned with the reassignments based on the Cytb data and are not discussed further.
Seven of the 16 species recognised with COI data (Fig. 2A) include representatives of other nominal species now to be recognised as synonyms.One, the ZooKeys 1204: 199-222 (2024)    P. krempfi clade includes 12 specimens of three nominal species currently assigned to two genera: P. krempfi, Hypsibarbus annamensis, and H. macrosquamatus.The newly collected specimens of all three nominal taxa were collected from their type localities in the Song Hong and northern Annam ecoregions and are embedded with P. krempfi specimens from the Song Hong with a mean similarity of 98.8% (± 0.29) (Fig. 2A).This clade received 83% bootstrap support.

P. hathe
Hypsibarbus annamensis, and H. macrosquamatus are synonyms of P. krempfi.The P. krempfi lineage in the Song Hong ecoregion has a COI sequence similarity of 97% (± 1.00) with P. alloiopleurus from the Song Hong, P. opisthopterus from the Upper Salween, P. burtoni from the Sittang -Irrawaddy, and P. schanicus from the Inle Lake, Myanmar (Table 2).The P. alloiopleurus clade (Fig. 2A, B) consists of 13 specimens of six nominal species (plus specimens labelled P. huangchuchieni in Wu et al. (2013) and Zheng et al. (2016)), currently in two genera: P. alloiopleurus, P. baolacensis, P. brevispinus, P. rhomboides, Acrossocheilus macrophthalmus, and A. xamensis collected in the Song Hong and northern Annam ecoregions from their type drainages (Fig. 2A).The newly collected specimens are embedded with P. alloiopleurus specimens from the Song Hong and show a mean similarity of 99.5% (± 0.20).This clade receives 100% bootstrap support.Thus, we conclude that P. baolacensis, P. brevispinus, P. rhomboides, A. macrophthalmus and A. xamensis are synonyms of P. alloiopleurus.The P. alloiopleurus lineage observed in the Song Hong ecoregion has a COI sequence similarity of 97% (±1.00) with P. krempfi from the Song Hong, P. huangchuchieni and P. laoensis from the Lower Lancang, and P. opisthopterus from the Upper Salween (Table 2).
The P. huangchuchieni clade consists of three specimens of two nominal species, P. huangchuchieni and P. angustus collected from their type drainages in the Lower Lancang ecoregion.These specimens have a mean similarity of 100% (± 0.00) (Fig. 2A).Poropuntius angustus is a synonym of P. huangchuchieni.The P. huangchuchieni lineage in the Lower Lancang ecoregion has a COI sequence similarity of 97% (± 1.00) with P. laoensis from the Lower Lancang, P. alloiopleurus from the Song Hong, and P. opisthopterus from the Upper Salween (Table 2).
The P. laoensis clade consists of 17 specimens of three nominal species, P. laoensis, P. aluoiensis, and P. bantamensis collected in the Lower Lancang, Kratie -Stung Treng, northern Annam, Chao Phraya, and Middle & Lower Salween ecoregions including from their type localities.The newly collected specimens are embedded with P. laoensis specimens from the Kratie -Stung Treng

Taxonomic conclusions
Based on molecular data, 17 nominal species of Poropuntius, Hypsibarbus, and Acrossocheilus are reduced to synonyms of the six valid species of Poropuntius listed below.Also considered a junior synonym of P. deauratus is P. consternans, referred to as a variant of P. bolovenensis by Kottelat (2013) in an environmental assessment of a hydroelectric power project in the Bolaven Plateau, Laos and relegated to the synonymy of P. deauratus by Kang et al. (2016).
Morphological and molecular data also provide evidence that newly collected specimens from the Annam ecoregion represent a new species of Poropuntius, described below.Synonyms listed are only those addressed in this study.Notes.Specimens of Poropuntius krempfi (Fig. 3A, B) were recovered in a clade including topotypic material of Barbus annamensis and Lissochilus macrosquamatus.The clade was sister to a large clade including P. alloiopleurus, P. heterolepidotus, P. huangchuchieni, P. laoensis, and P. opisthopterus (Fig. 2A).Specimens of P. krempfi from northern Annam (MNHN 1935:0337, MNHN 1969:42) were assigned by Rainboth (1996) to Hypsibarbus annamensis presumably due to the presence of a few tiny tubercles on the tip of the snout, but this trait is not useful for separating all species of the two genera.Some of our 41 specimens of P. krempfi have tiny tubercles on the tip of the snout.Rainboth (1996) noted that this species was unusual in being the only species of Hypsibarbus on the coastal side of the Annam Cordillera and in having a much longer dorsal spine with 26-28 serrations, compared to 20 or fewer in other species in the Mekong River drainage.

Taxonomic account
Hypsibarbus macrosquamatus from Song Hong, northern Vietnam (type: DVZUT) described by Mai (1978) and treated as Acrossocheilus macrosquamatus by Nguyễn and Ngô (2001) resembles our P. krempfi specimens from northern Annam with a lateral-line scale count of 29-33.
Distribution.Poropuntius krempfi is found in the Song Hong and northern Annam ecoregions.
According to Nguyễn (2001), A. macrophthalmus resembles P. alloiopleurus but has an eye diameter longer than the snout.Our 12 specimens of P. alloiopleurus have eye diameters that are shorter or longer than the snout.
Nguyễn and Ngô (2001) distinguished P. baolacensis from P. brevispinus in having 28-30 vs 18-22 serrae along the posterior margin of the last simple dorsal-fin ray.Our 12 specimens of P. alloiopleurus have 20-29 serrae on the last dorsal-fin ray, partially overlapping the serrae counts given to distinguish P. baolacensis and P. brevispinus.
Distribution.Poropuntius alloiopleurus is found in the Song Hong ecoregion.Notes.In the original description of P. angustus, Kottelat (2000) made no specific mention of P. huangchuchieni.Topotypic material (UNS 2018-07-11, Fig. 3F) from the Nam Nứa, Lower Lancang ecoregion, in the upper reach of Nam Ou, the type locality of P. angustus, was genetically identical to that of P. huangchuchieni from the Lancang Jiang in Yunnan.Distribution.Poropuntius huangchuchieni is found in the Lower Lancang ecoregion.
Poropuntius deauratus occurs over a large geographic area and shows considerable variation in shape and color (Fig. 4B-H) as well as in the structural traits related to feeding morphology.
Distribution.Poropuntius deauratus is found in the Western Malaysia, Malay Peninsula Eastern Slope, Eastern Gulf of Thailand, Mekong Delta, Kratie-Stung Treng, Khorat, northern Annam, and southern Annam ecoregions.Diagnosis.Poropuntius anlaoensis is the only species of the genus found on the coastal side of the Annamite Cordillera and nowhere else.It differs from all other species of Poropuntius genetically (Fig. 2A), and by having distal margin of dorsal fin distinctly concave (vs straight to slightly concave).It most closely resembles P. deauratus but has 29-31 (vs 25-28) lateral-line scales, snout distinctly pointed (vs slightly pointed), and caudal fin light yellow with bold black submarginal stripes (vs bright lemon yellow to dusky with bold to faint black submarginal stripes).Poropuntius anlaoensis differs from P. genyognathus, P. hampaloides, and P. melanogrammus in having barbels (vs no barbels); from P. heterolepidotus and P. hathe in having scales on posterior half of body not markedly smaller (vs markedly smaller) than those on anterior half; from P. alloiopleurus, P. burtoni, P. carinatus, P. huangchuchieni, P. kontumensis, P. krempfi, P. laoensis, P. opisthoptera, and P. schanicus in having 29-31 (vs > 32) lateral-line scales and bold black submarginal stripes (vs no bold black stripes) on caudal fin.

Poropuntius anlaoensis
Description.General appearance in Fig. 5; meristic and morphometric data of 11 specimens in Table 3. Head conical, longer than deep, depth 1.2-1.4× in HL.Snout pointed.Tubercles tiny and few on tip of the snout, many irregular transverse rows of small tubercles reaching front of eyes in male.Mouth subterminal and oblique, extending posteriorly in length slightly longer than eye diameter and broadly horseshoe-shaped (Fig. 5C).Rostral barbel shorter than maxillary barbel, both longer than eye diameter.
Body moderately deep and compressed, depth approximately 2.8-3.3 × in SL.Dorsal body profile convex, slightly convexity from nape with narrow dorsum almost straight in front of dorsal origin to dorsal fin.Base dorsal fin decreasing in height nearly straight dorsal margin of the body, extending from dorsal-fin origin to narrowest part of the caudal peduncle.Ventral profile rounded, rising through anal-fin insertion to caudal-fin base.Caudal peduncle slender, moderately shallow and long, 1.6-2.1 × longer than deep.Anus immediately in front of anal fin.Lateral line complete, 29-31 scales; 10-12 predorsal scales; 5/1/3 scales in transverse row anterior to pelvic-fin origin.Lateral-line tubes extending at least halfway across each scale, with accessory pore on ventral branch on nearly every lateral-line scale.Dorsal iv-8.5, pectoral i-15, pelvic i-8, and anal iii-5.5.
Dorsal fin high and sharply pointed at apex, last unbranched ray longest, followed by first branched ray which is considerably shorter.Last unbranched ray ossified with 18-20 serrae.Posterior extensions of serrae forms straight

Discussion
The objective of this study was to test, using molecular data, the species diversity of Poropuntius, especially that of Vietnam in which 15 species names have been recognised recently as valid.As noted above, most species of Poropuntius have been described using only morphological data, and recent investigations using molecular data suggested that some morphological hypotheses were incorrect.Our data indicate that 17 names assigned to species are synonyms.Several factors led to the inflation of species names for Poropuntius.These include limited efforts overall in collecting and classifying cyprinid fishes, especially in northern Vietnam, limited access to type specimens in European museums, limited availability of publications (often in foreign languages -although now this is being alleviated by online access), inadequate descriptions containing limited information, and failure to appreciate morphological variation related to trophic diversification within species.Finally, a substantial portion of the cyprinid diversity of northern Vietnam is shared with that of southern China, but researchers in the two countries have not had access to specimens from both countries or to original descriptions in other languages.Consequently, studies were limited to in-country fauna, and species in each country were treated as endemic to that country.
Ecomorphological variation in Poropuntius was described and figured by Roberts (1998: figs 6, 8), and Kang et al. (2016) interpreted P. bolovenensis, P. lobocheiloides, and P. solitus as ecomorphs of P. bolovenensis.The often-extreme intraspecific phenotypic variation in the shape of the mouth and lips, and in the development of the horny sheath on the lower jaw, has led to taxonomic confusion in Poropuntius.Ecophenotypic variation is expressed especially clearly in species occupying pools isolated by barriers such as waterfalls in streams on plateaus at high elevations (e.g., on the Bolaven, Kontum, and Langbiang Plateaus) and in drainages leading to the Gulf of Tonkin (in the Song Hong and Annam ecoregions).Species not exhibiting such extreme polymorphism may nevertheless carry information in their genomes necessary to generate an array of potentially taxonomically confusing, continuous variation as well as more disjunct phenotypes.For example, the position of the dorsal fin, often used in taxonomic diagnoses, varies from more anteriorly to more posteriorly positioned in Poropuntius, as seen in P. alloiopleurus, P. huangchuchieni, and P. laoensis (Wu et al. 2013;this study).Specimens of Poropuntius from flowing water habitats are characterised by a more elongated body and a relatively long caudal peduncle, while those from pools have a deeper body and larger unpaired fins (Figs 3A,B,D,E,4G,H).Similar patterns have been described in other cyprinids, e.g., Rasbora paviana and Lobocheilos rhabdoura, with populations inhabiting fast-flowing streams having more slender bodies than those inhabiting slower-flowing habitats (Stolbunov et al. 2011;Ciccotto and Page 2016).
During a decade of field work on Southeast Asian freshwater fishes, the first author and colleagues have collected samples of P. deauratus, P. krempfi, P. alloiopleurus, and P. laoensis revealing marked intraspecific phenotypic variation.These samples include those from river basins in which few or no fish collections have been made or reported upon previously, and which fill in gaps in the distributions of these species.Pronounced variation among adult individuals in these species has been observed in body depth, dorsal-fin spine length, ossification and serration of the last simple dorsal-fin ray, body colour, and caudal fin colour.For example, in the wide-ranging P. deauratus, the typical colour in life is a silvery to light green gold body with bright yellow on the posterior half of the body and a bright lemon-yellow caudal fin with bold black submarginal stripes.However, colour in this species may be much more subtle with the body uniformly dusky to dark and the fins dusky (Fig. 4B, H).

Conclusions
This study has revealed that scientific names have been applied erroneously to populations of P. krempfi, P. alloiopleurus, P. huangchuchieni, P. laoensis, P. kontumensis, and P. deauratus.Errors in the taxonomy of Poropuntius have resulted primarily from inadequate sampling and reliance on characters that vary intraspecifically.Additional samples from unsampled or poorly sampled populations allowed the use of molecular data to test previous species hypotheses.More sampling and use of molecular as well as morphological data on species of Poropuntius that could not be included in this study are likely to require additional taxonomic changes.

Figure 1 .
Figure 1.Sampling sites of Poropuntius in this study in 15 freshwater ecoregions shown in various colours.

Figure 2 .
Figure 2. Maximum-likelihood tree based on (A) COI and (B) Cytb mitochondrial gene sequences for species of Poropuntius.Numbers on branches are ML bootstrap values (values > 50% shown).Bold sample labels are sequences from type localities.Blue sample labels in the Cytb tree are sequences from Wu et al. (2013).Colours of clades correspond to the freshwater ecoregions in Fig. 1.

Figure 5 .
Figure 5. Poropuntius anlaoensis A adult holotype (UNS00762, 139.46 mm SL) in life B in preservative C ventral view of head D dorsal view.Scale bars: 10 mm.

Table 1 .
Cytochrome b genetic distances between species in the phylogenetic analysis with P. genyognathus as the outgroup using the Kimura 2-parameter model, standard error estimates shown above the diagonal with bootstraps 1000.Poropuntius normani, P. bolovenensis, P. lobocheiloides, P. smedleyi, and P. solitus are synonyms of P. deauratus.The P. deauratus lineage in the Eastern Gulf of Thailand drainages, Malay Peninsula, Mekong Delta, Kratie -Stung Treng, Khorat Plateau, and Annam ecoregions has a COI sequence similarity of 96% (± 1.00) with P. carinatus from the Lower Lancang and P. kontumensis from the Kratie -Stung Treng ecoregion (Table 2).

deauratus (Valenciennes, in Cuvier & Valenciennes, 1842)
) were collected in the upper Xekong drainage of Central Vietnam, the type locality, i.e., in the Lao Mountains of Cochin-China, now Central Vietnam.Topotypic material of P. bantamensis and P. aluoiensis resembled P. laoensis in having the proximal half of the caudal fin orange and the distal half bright yellow, and bold black submarginal stripes on the caudal fin.Distribution.Poropuntius laoensis occurs in the Lower Salween, Chao Phraya, Lower Lancang, northern Annam, and Kratie-Stung Treng ecoregions.Poropuntius smedleyide Beaufort, 1933: 44.Type locality: Malaysia, Johor.