Forgotten for Decades: Revalidation and Redescription of Raiamas harmandi (Sauvage, 1880) (Cypriniformes: Danionidae) from the Mekong River Basin

Abstract

The genus Raiamas currently comprises 18 valid species, only 2 of which occur in Asia; the remaining 16 are endemic to Africa. Raiamas harmandi was originally described by Sauvage in 1880 as Bola harmandi, which is distributed in the Great Lakes, Cambodia, the Mekong River Basin. It was considered a synonym of R. guttatus by later researchers. In this study, we examined 49 Raiamas individuals from the Mekong, Irrawaddy, and Salween river basins, recording both meristic counts and morphometric measurements. Based on the morphological evidence, we revised the taxonomy of Raiamas in the Mekong River Basin, confirming R. harmandi as a valid species and providing a comprehensive redescription. Raiamas harmandi can be distinguished from R. guttatus mainly by having more predorsal scales (25–28 vs. 21–23) and a different color pattern on the lateral body. Utilizing a total of 44 aligned COI and Cyt b sequences—including eight newly sequenced individuals of Raiamas from three river basins—we reconstructed its phylogenetic relationships. The analysis strongly supported four R. harmandi individuals from the Mekong River Basin forming a distinct clade, which was the sister to the clade comprising five R. guttatus individuals from the Irrawaddy and Salween river basins. Genetic distances between R. harmandi and R. guttatus ranged from 14.0 to 14.9% for COI and 16.1 to 17.0% for Cyt b. Distributionally, R. harmandi occurs throughout the Mekong River Basin, as evidenced by combined voucher specimens and molecular sequence data.

1. Introduction

The family Danionidae, with 367 species in 39 genera, is currently recognized as comprising 4 subfamilies: Danioninae, Chedrinae, Esominae, and Rasborinae [1,2,3]. Danioninae, Esominae, and Rasborinae exhibit distributions restricted to South, Southeast, and East Asia (with Rasbora steineri constituting the only species occurring in Southeast China), whereas Chedrinae occurs across both Asia and Africa. The genus Raiamas Jordan, 1919, one of the most elongate-bodied genera within Chedrinae and the entire Danionidae, inhabits freshwater rivers throughout Africa, South Asia, and Southeast Asia, characterized most notably by an exceptionally elongate gape [4,5].

With 18 currently valid species [1], Raiamas represents a relatively low species diversity genus within Danionidae. Sixteen species are endemic to Africa, where no new taxa have been described since the addition of two species in 2018 [5]. In contrast, Asia hosts only two species and has seen no new species of Raiamas for over a century. In earlier taxonomic studies, numerous African species were assigned to the genus Raiamas owing to their morphological similarity, particularly the elongate maxilla reaching to or beyond the eye center and the lateral body usually marked with numerous spots or black bars—features that distinguish Raiamas from other members of the family Danionidae [5]. However, recent evidence indicates that the two Asian species, R. bola (Hamilton, 1822) and R. guttatus (Day, 1870), differ from their African congeners in the presence of barbels, the relative length of the upper jaw, the color pattern of the caudal fin, and the number of branched anal-fin rays [6,7,8,9], as well as in their phylogenetic relationships [10]. Taken together, these findings suggest that the African taxa may represent a distinct genus.

Raiamas bola was originally described as Cyprinus bola by Hamilton (1822) based on specimens from the Brahmaputra River, India [6]. Subsequently, several new Indian taxa were described, including C. goha, Opsarius gracilis, O. megastomus, Leuciscus salmoides, Brailius corbetti, and B. jalkapoorei [6,11,12,13,14]. These taxa later were synonymized with R. bola through taxonomic revision [15,16,17]. Raiamas bola is now considered widely distributed along the Himalayan foothills of South Asia, encompassing Pakistan, Nepal, Bhutan, India, and Bangladesh.

Raiamas guttatus was first described as Opsarius guttatus by Day (1870) based on collections from the Irrawaddy River, Myanmar [7]. It was later recorded in the Salween River Basin. Sauvage described Bola harmandi in 1880 from the Great Lake (Tonlé Sap Lake, the Mekong River Basin) of Cambodia [18], while Yang and Hwang described Luciosoma fasciata in 1964 from the Lancangjiang River (the upper Mekong River Basin) in Yunnan Province, China [19]. Subsequent revisions synonymized both Mekong River Basin taxa under R. guttatus [15,20]. Recent records from Sumatra and the Malay Peninsula have expanded its known range [21,22], resulting in the current interpretation that nearly all Southeast Asian Raiamas populations represent R. guttatus. However, neither morphological nor phylogenetic studies have sufficiently considered implications of this broad trans-basin distribution. Investigations have typically relied on material from single river system, potentially obscuring hidden diversity within this widely distributed species.

The aim of this study is to employ comparative morphology and phylogenetic reconstruction on R. guttatus samples from the Irrawaddy, Salween, and Mekong River basins. The study seeks to assess species-level divergence among these populations, with the goal of determining whether the Mekong populations constitute a distinct and valid species. Based on the findings, we aim to revalidate Raiamas harmandi (originally described as Bola harmandi) and provide a full redescription herein.

2. Materials and Methods

2.1. Sample Collection and Morphological Analysis

A total of 49 specimens of Raiamas were morphologically examined in this study (Figure 1). Among them, 45 specimens were the previous voucher specimens of the Muséum National d′Histoire Naturelle, Paris, France (MNHN); the Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China (KIZ); and the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China (IHB). In addition, two specimens of R. harmandi and two specimens of R. guttatus were recently collected in Yunnan Province for this study. Tissues of pectoral fins from the collected specimens were cut and fixed with 95% ethanol for subsequent DNA extraction. After that, the collected specimens were fixed in 10% formalin solution and transferred to 75% alcohol solution for long-term preservation in the Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China (

Table 1. Information on COI and Cyt b sequences used in phylogenetic analysis. ¹ denotes Asian endemic species; ² denotes African endemic species. * indicates individuals analyzed for both morphology and DNA sequences.

Species	Voucher No.	COI	Cyt b	Source
Ingroup
Barilius cf. barila 1	CTOL02812	HM224138	HM224257	[10]
Barilius vagra 1	CTOL03301	HM224140	HM224259	[10]
Chelaethiops bibie 2	CTOL02657	HM224141	HM224260	[10]
Chelaethiops bibie 2	CTOL03164	HM224142	HM224261	[10]
Chelaethiops elongatus 2	AMNH-I-247817	JX196996	JX197006	[8]
Engraulicypris Sardella 2	AMNH-I-248841	JX196997	JX197007	[8]
Engraulicypris brevianalis 2	CTOL03497	HM224176	HM224295	[10]
Leptocypris modestus 2	AMNH-I-240469	JX196998	JX197008	[8]
Leptocypris niloticus 2	CTOL02619	HM224174	HM224293	[10]
Leptocypris niloticus 2	CTOL03165	HM224175	HM224294	[10]
Opsaridium boweni 2	AMNH-I-243665	JX197000	JX197009	[8]
Opsaridium ubangiense 2	CTOL03214	HM224193	HM224312	[10]
Raiamas batesii 2	AMNH-I-249530	JX197002	JX197010	[8]
Raiamas bola 1	CTOL03200	HM224212	HM224329	[10]
Raiamas buchholzi 2	CTOL03213	HM224213	HM224330	[10]
Raiamas buchholzi 2	no6265	AP012118	AP012118	Direct Submission
Raiamas christyi 2	AMNH-I-250553	JX197003	JX197011	[8]
Raiamas guttatus 1*	KIZ2025000152	PV960713	PV976866	This study
Raiamas guttatus 1*	KIZ2025000153	PV960714	PV976867	This study
Raiamas guttatus 1	CXY20190499	PV960710	PV976868	This study
Raiamas guttatus 1	PZ20180114	PV960711	PV976869	This study
Raiamas guttatus 1	QT20180008	PV960712	PV976870	This study
Raiamas harmandi 1	QT20200210	PV960707	PV976864	This study
Raiamas harmandi 1*	KIZ2025000151	PV960709	PV976863	This study
Raiamas harmandi 1	XP0901418	PV960708	PV976865	This study
Raiamas harmandi 1	MNHN-2012-0078	JQ346159	JQ346143	[23]
Raiamas salmolucius 2	AMNH-I-251147	JX197004	JX197012	[8]
Raiamas senegalensis 2	CTOL02658	HM224215	HM224332	[10]
Raiamas senegalensis 2	CTOL03171	HM224216	HM224333	[10]
Raiamas senegalensis 2	N/A	AP010780	AP010780	[24]
Raiamas steindachneri 2	CK473	AP012113	AP012113	Direct Submission
Rastrineobola argentea 2	AMNH-I-256386	JX197005	JX197013	[8]
Luciosoma bleekeri 1	CBM-ZF-11202	AP011399	AP011399	[10]
Luciosoma setigerum 1	CBM-ZF-11273	AP011423	AP011423	[10]
Salmostoma bacaila 1	CBM-ZF-11516	AP011223	AP011223	[10]
Cabdio morar 1	CBM-ZF-11391	AP011335	AP011335	[10]
Malayochela maassi 1	LR1640	FJ753486	EF151098	[25]
Nematabramis steindachneri 1	LR1654	FJ753496	EF151106	[25]
Neobola bottegoi 2	CTOL02623	HM224178	HM224296	[10]
Opsarius pulchellus 1	N/A	MW625808	MW625808	[26]
Opsarius caudiocellatus 1	N/A	OM617729	OM617729	[27]
Securicula gora 1	CTOL03489	HM224250	HM224381	[10]
Outgroup
Danio rerio	N/A	KT624625	KT624625	[28]
Danio rerio	N/A	KT624626	KT624626	[28]

).

We mainly follow Stiassny et al. [29] and Manda et al. [5] in studying the morphology of Raiamas. In particular, 25 measurements and 12 meristic counts were processed on the left side of each specimen, except in cases where damage necessitated the use of the right side. Measurements were taken point to point with a digital caliper to 0.1 mm and given as percentage of standard length (SL), except for subunits of the head, which were given as percentage of head length (HL). Counts of dorsal-fin rays, anal-fin rays, abdominal vertebrae (excluding the four vertebrae of the Weberian complex), and caudal vertebrae were made on radiographs with the KUBTEC XPERT 80-L X-ray System. The last two branched rays, articulating on a single pterygiophore in the dorsal and anal fins, were counted as one. Caudal vertebrae were counted from the first vertebra with a hemal spine articulating with the first anal-fin pterygiophore to the first preural centrum. The first preural centrum was counted as one. The count of lateral-line scales excluded the scales on the caudal fin. Transverse scale counts included scales above the lateral line (lateral line to dorsal-fin origin) and scales below the lateral line (lateral line to pelvic-fin origin), and the scale immediately in front of the dorsal fin was counted as ½. The predorsal scale count included all the scales on the dorsal midline in front of the dorsal-fin origin [20].

2.2. DNA Extraction, PCR Amplification, and Sequencing

In the present study, eight tissue samples of Raiamas from three river basins in three countries were selected for DNA extraction (Figure 1; Table 1). Total genomic DNA was extracted from ethanol-fixed fin tissues using the FastPure Cell/Tissue DNA Isolation Mini Kit-BOX (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s protocol. Two mitochondrial gene markers, cytochrome c oxidase subunit I (COI) and cytochrome b (Cyt b), were amplified using polymerase chain reaction (PCR) by the following primers: FishFl: 5′-TCAACCAACCACAAAGACATTGGCAC-3′ and FishR1: 5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′ (for COI); LA-danio: 5′-GACTYGAARAACCACYGTTG-3′ and HA-danio: 5′-CTCCGATCTTCGGATTACAAG-3′ (for Cyt b) [30,31]. PCRs were performed in 30 μL total volume: 2 μL of DNA template, 2 μL of each primer, 15 μL of Taq PCR Mix (Sangon Biotech Co., Ltd., Shanghai, China), and 9 μL of ddH₂O. The PCR conditions were as follows. For COI: 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; then, 72 °C for 10 min. For Cyt b: 95 °C for 2 min; 35 cycles of 95 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min; then, 72 °C for 10 min. The PCR products were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The sequencing results were assembled and corrected using SeqMan Pro 11.1.0 [32].

2.3. Phylogenetic Analysis and Genetic Distance

For phylogenetic analysis, we selected 42 individuals of 30 species from the subfamily Chedrinae as ingroups, of which the sequences of 8 individuals were from this study and the remaining sequences were downloaded from NCBI (Table 1). Sequences from two individuals of Danio rerio were chosen as outgroups (Table 1). Implemented in the PhyloSuite 1.2.3 [33,34], sequences of COI and Cyt b genes were separately aligned with MAFFT 7.505 [35,36], trimmed with the automated alignment trimming tool trimAl 1.2 [37], and then concatenated. The best partitioning schemes and substitution models were estimated using PartitionFinder 2.1.1 [38], and the Bayesian Information Criterion (BIC) was applied. The maximum-likelihood (ML) tree was inferred with IQ-TREE 2.2.0 [39] under the GTR+I+G model. Support values were estimated through 5000 ultrafast bootstrap replicates [40]. The Bayesian inference (BI) tree was constructed with MrBayes 3.2.7 [41]. Four independent runs were performed with 50 million generations of Monte Carlo Markov chain (MCMC), sampling every 1000 generations. The first 25% of MCMCs were discarded as burn-in. Convergence of four runs was checked in Tracer 1.7.1 [42]. All effective sample size (ESS) values were above 200. Trees were visualized and optimized in iTOL 7.2 webserver [43]. The pairwise genetic distances for aligned sequences of COI and Cyt b among each species were calculated using MEGA X 10.2.2 under the pairwise Kimura two-parameter (K2P) model with 1000 bootstrap replicas [44,45].

3. Results

3.1. Molecular Phylogeny and Genetic Distance

Phylogenetic reconstruction incorporated 44 aligned sequences (2 outgroup, 42 from subfamily Chedrinae) (Figure 2; Table 1), including five Raiamas guttatus and three R. harmandi specimens sequenced in this study, supplemented by one GenBank-deposited Raiamas individual from Laos′s Mekong River Basin. In general, the ML and BI trees shared a similar topology (Figure 2). The results of the phylogenetic study showed that all the Raiamas materials from Asia clustered as a monophyletic group, and four individuals of R. harmandi in the Mekong River Basin (including the individual from Laos) form an independent lineage on the phylogenetic tree, with sister groups of R. guttatus in the Irrawaddy and Salween river basins. This result was highly supported by both BI and ML analyses (maximum-likelihood bootstrap values, BP = 97; Bayesian posterior probabilities, PP = 1). The clade consisting of R. harmandi and R. guttatus first clustered with R. bola and subsequently clustered with an African group comprising several Raiamas species and allied genera (Figure 2). However, these relationships received only low support values throughout the topology.

The interspecific genetic distances for COI and Cyt b sequences based on the K2P model between R. harmandi and its closest congener R. guttatus are 14.0–14.9% and 16.1–17.0%, respectively (

Table 2. Genetic distances of the COI and Cyt b genes among three Asian Raiamas species.

	COI				Cyt b
Species	Intraspecific	1	2	3	Intraspecific	1	2	3
1 Raiamas harmandi	0–0.005	–	–	–	0–0.009	–	–	–
2 Raiamas guttatus	0–0.003	0.140–0.149	–	–	0–0.009	0.161–0.170	–	–
3 Raiamas bola	–	0.163–0.165	0.162–0.164	–	–	0.200–0.211	0.214–0.216	–

). The intraspecific genetic distances in R. harmandi are 0–0.5% for COI and 0–0.9% for Cyt b (Table 2), which are much lower than the interspecific genetic distances.

3.2. Taxonomy and Morphology

Raiamas harmandi stat. rev. (Sauvage, 1880) (Figure 3, Figure 4a and Figure 5 and Figure 6a,c;

Table 3. Body proportions and counts data of Raiamas harmandi (n = 29, except vertebral counts where n = 23) and R. guttatus (n = 20).

Characters	Raiamas harmandi			R. guttatus
	Holotype	Mean ± SD	Range	Mean ± SD	Range
Standard length (SL, mm)	160.0	167.4 ± 45.2	88.6–240.2	158.8 ± 28.0	121.7–210.6
In Percent of SL (%)
Body depth	21.1	20.9 ± 2.4	16.9–26.2	22.4 ± 2.1	17.4–26.6
Pectoral-fin length	19.4	18.0 ± 1.0	16.3–20.0	19.0 ± 0.9	17.2–20.6
Prepectoral length	28.4	27.3 ± 0.9	25.6–29.4	28.1 ± 1.2	25.8–30.4
Pelvic-fin length	14.1	13.0 ± 0.8	11.9–14.3	12.6 ± 0.6	11.6–14.4
Prepelvic length	49.8	50.4 ± 1.6	45.6–53.6	51.4 ± 1.3	49.6–54.6
Dorsal-fin length	16.0	17.6 ± 0.8	16.0–19.0	16.6 ± 0.7	15.3–17.6
Dorsal-fin base length	10.8	10.9 ± 0.7	9.8–12.2	10.6 ± 0.6	9.6–11.6
Predorsal length	57.9	56.8 ± 1.6	51.3–59.4	57.7 ± 1.3	54.3–60.1
Anal-fin length 1	14.8	14.6 ± 0.9	13.0–16.5	14.5 ± 0.6	12.8–15.3
Anal-fin length 2	4.6	5.7 ± 0.6	4.3–6.7	5.2 ± 0.5	4.1–6.2
Anal-fin base length	13.4	13.4 ± 0.6	12.1–14.5	15.1 ± 1.1	13.2–17.2
Preanal length	70.0	70.4 ± 2.2	64.6–74.5	71.9 ± 1.7	69.2–76.2
Caudal peduncle length	17.9	17.2 ± 1.0	15.5–19.4	16.7 ± 1.1	13.9–18.7
Caudal peduncle depth	9.6	8.7 ± 0.5	7.9–9.8	9.2 ± 0.4	8.2–9.8
Head length (HL)	27.8	27.0 ± 0.9	25.4–29.2	27.5 ± 1.1	25.8–29.7
In Percent of HL (%)
Head depth	61.5	61.5 ± 2.0	57.1–66.6	60.6 ± 2.4	56.6–65.0
Head width	41.9	41.9 ± 2.3	38.8–48.7	44.1 ± 3.1	38.3–50.0
Snout length	26.8	28.7 ± 1.9	24.7–33.4	28.7 ± 1.5	25.7–31.7
Interorbital width	26.8	25.9 ± 1.3	23.6–28.2	26.7 ± 1.1	23.2–28.2
Upper-jaw length	67.1	64.2 ± 2.5	57.9–68.6	59.2 ± 1.3	56.3–61.9
Eye diameter	18.2	17.4 ± 2.2	13.9–22.1	17.0 ± 1.7	14.1–20.4
Distance between nostril and snout tip	18.9	19.9 ± 1.7	16.7–24.6	20.0 ± 1.3	18.0–23.3
Horizontal eye to operculum distance	29.7	32.5 ± 2.3	28.2–37.1	33.5 ± 2.3	28.4–38.1
Opercular length	24.8	20.8 ± 1.5	18.2–24.8	22.0 ± 1.4	19.4–24.3
Counts	Holotype	Median	Range	Median	Range
Total number of vertebrae	42	43	41–44	43.5	42–44
Abdominal vertebrae	22	22	21–23	22	21–23
Caudal vertebrae	20	21	20–22	21	21–22
Dorsal-fin rays	iii, 7	iii, 7	iii, 7	iii, 7	iii, 7
Anal-fin rays	iii, 10	iii, 10	iii, 10–iii, 11	iii, 11	iii, 10–iii, 12
Pectoral-fin rays	i, 15	i, 14	i, 13–i, 15	i, 13.5	i, 13–i, 14
Pelvic-fin rays	i, 8	i, 8	i, 8	i, 8	i, 8
Lateral line scales	45	47	45–50	48	46–50
Lateral line–dorsal-fin origin scales	9½	9½	8½–9½	9½	8½–9½
Lateral line–pelvic-fin origin scales	3	3	3–4	3	3–4
Predorsal scales	25	26	25–28	22	21–23
Circumpeduncular scales	19	18	18–20	18	18–19

).

Bola harmandi Sauvage, 1880: 231 [18] (original description, type locality: Great Lakes, Cambodia).

Barilius guttatus: Smith, 1945: 152 [46] (Chieng Sen, Mekong River baisn, Thailand); Li, 1976: 117 [47] (Jinghong, Mengla, Xishuangbanna, Yunnan Province, China).

Luciosoma fasciata Yang & Hwang, 1964: 53 [19] (Yunjinghong, Xishuangbanna, Yunnan Province, China).

Bola guttatus: Kottelat, 1984: 797 [48] (short review of the holotype of Bola harmandi).

Raiamas guttatus: Kuang, in Chu & Chen, 1989: 23 [49] (Mengla, Lancang, Jinghong, Mekong River Basin, Yunnan Province, China); Rainboth, 1996: 70 [50] (Cambodia); Menon, 1999: 24 [16] (list); Kottelat, 2001: 76 [20] (list); Kottelat, 2013: 150 [15] (list).

Holotype. MNHN-IC-A-2399 (Figure 3), 160.0 mm SL, Great Lakes (Tonlé Sap Lake), Cambodia.

Additional materials examined. KIZ 1973000965, 193.8 mm SL, Menglang Town, Pu′er, Yunnan, China, 1973; KIZ 1986000898–904, 1988001728, 184.1–233.9 mm SL, Mengla County, Xishuangbanna, Yunnan, China, April 1983; KIZ 1986002060, 191.1 mm SL, Mengla County, Xishuangbanna, Yunnan, China, 1986; KIZ 1996001371, 1996001373–1375, 1996001377, 88.6–137.6 mm SL, Mengla County, Xishuangbanna, Yunnan, China, 10 June 1996; KIZ 2008000171–172, 144.6–239.9 mm SL, Nanla River, Daluo Town, Xishuangbanna, Yunnan, China, 26 January 2008; KIZ 2012001350–1351, 181.2–190.3 mm SL, Menglun Town, Xishuangbanna, Yunnan, China, 25 April 2012; KIZ 2012001405, 237.4 mm SL, Mengla County, Xishuangbanna, Yunnan, China, 26 April 2012; KIZ 2025000150–151, 176.5–240.2 mm SL, 21.8881° N, 101.2740° E, Luosuojiang River, Menglun Town, Xishuangbanna, Yunnan, China, 29 March 2025 (Figure 4a); IHB 584078, 584147, 584201, 584213, 584216, 584228, 94.6–153.5 mm SL, Yunjinghong, Xishuangbanna, Yunnan, China, 1958.

Comparative materials. Raiamas guttatus: Irrawaddy River Basin: KIZ 1978000884–885, 191.7–208.5 mm SL, Nabang Town, Dehong, Yunnan, China, 1978; KIZ 2025000152, 159.2 mm SL, 24.7288° N, 97.5653° E, Mengnai River, Nabang Town, Dehong, Yunnan, China, 28 October 2024 (Figure 4b). Salween River Basin: KIZ 2002001879, 165.1 mm SL, Baishitou River, Yongde County, Lincang, Yunnan, China, June 2002; KIZ 2002002170–2171, 178.8–197.1 mm SL, Nanting River, Yongde County, Lincang, Yunnan, China, 18 June 2002; KIZ 2010002509–2510, 152.2–162.1 mm SL, Damengtong River, Changning County, Baoshan, Yunnan, China, 26 April 2010; KIZ 2015000651–653, 126.2–210.6 mm SL, Nanpeng Village, Gengma County, Lincang, Yunnan, China, 6 February 2015; KIZ 2015005462, 2015005468–69, 2015005481, 2015005483, 2015005490, 2015005493, 2015005496, 121.7–185.7 mm SL, Nanting River, Gengma County, Lincang, Yunnan, China, 4 June 2015; KIZ 2025000153, 128.9 mm SL, 24.5653° N, 99.0436° E, Nujiang River, Longling County, Baoshan, 11 May 2024 (Figure 4c).

Diagnosis. Raiamas harmandi can be distinguished from all other Asian Raiamas species by the following combination of characteristics: a distinctive color pattern of numerous small spots irregularly scattered on the lateral body; predorsal scales 25–28; lateral-line scales 45–50; total number of vertebrae 41–44; caudal vertebrae 20–22. It can be further distinguished from the most similar species, R. guttatus, by a proportionally longer upper jaw (57.9–68.6% HL vs. 56.3–61.9% HL).

Description. Meristic counts and morphometric measurements are listed in Table 3. Body elongate, compressed. Dorsal profile straight, slightly convex at dorsal-fin origin; ventral profile straight anteriorly, ascending at anal-fin base, straight posterior to anal fin. Caudal-peduncle length greater than its depth. Scales cycloid; scales on lateral body larger than dorsal/ventral scales; axillary scales present and developed at pectoral-fin and pelvic-fin bases (Figure 5a,b).

Lateral line complete, decurved ventrally, then straight to caudal-fin base; lateral-line scales 45–50; predorsal scales 25–28, irregularly arranged; circumpeduncular scales 18–20; scale rows between dorsal-fin origin and lateral line 8½–9½; scale rows between pelvic-fin origin and lateral line 3–4.

Head pointed, head length greater than body depth. Snout pointed. Mouth terminal, gape large; lower jaw slightly longer than upper jaw, posterior tip extending beyond posterior margin of orbit; anterior tip of lower jaw with prominent knob-like process fitting into upper-jaw notch (Figure 5c). Lips with weakly developed cuticular margins. Maxillary barbels minute, one pair (Figure 5d). Eyes small, dorsolaterally situated, closer to snout tip than to opercular margin. Interorbital width less than snout length, about twice eye diameter. Internarial region slightly concave.

Dorsal fin iii, 7 (29 specimens), insertion posterior to pelvic-fin origin, last ray tip extending posterior to anal-fin base; pectoral fin i, 13 (7); i, 14 (18); or i, 15 (4); subtriangular, tip extending beyond midpoint of pectoral–pelvic distance. Pelvic fin i, 8 (29), with truncate posterior margin, not reaching anal-fin origin; anus adjacent to anal-fin origin; anal fin iii, 10 (16) or iii, 11 (13), origin slightly posterior to dorsal-fin base, distance to pelvic-fin origin less than to caudal-fin base; caudal fin forked, upper and lower lobes subequal.

Coloration in life. Body generally grayish-green, back light brown. One row of large bluish-black spots on lateral body dorsally; 30–50 small, elliptical, bluish-black spots scattered irregularly below in adults. Several black spots between the upper jaw and eye. Orange on the bases and margins of pectoral, pelvic, and anal fins. Dorsal fin grayish-black with an orange base. Orange margins on both upper and lower lobes of the caudal fin. A black band extending posteriorly from the caudal peduncle through the middle of the fin to the tip of the lower lobe (Figure 6a,c).

Coloration in preservative. Fixed in formalin and briefly transferred in alcohol, the lateral and ventral body bright white, back grayish-black. Spots on the lateral body faded and light black. Pelvic and anal fins light white. Pectoral-fin margins light black. Dorsal fin grayish-black. Black band of caudal fin deep. With long-term preservation in alcohol, body dark brownish, back darker. Spots on sides; bars on dorsal and caudal fin. All fins light brownish (Figure 3 and Figure 4a).

Distribution. Raiamas harmandi is widely distributed in the Mekong River Basin, with records from China (the lower Lancang River), Laos, Vietnam, Thailand, and Cambodia (Figure 1). Species of the genus Raiamas have also been recorded in the Chao Phraya River Basin, Peninsular Malaysia, and South Sumatra, and it remains to be confirmed whether they are R. harmandi.

Habitat. Raiamas harmandi inhabits medium to large rivers, usually with strong and fast currents, and it feeds on small fish and aquatic insects [19,49,50]. Juveniles grow in small rivers or mountain streams.

Etymology. The name of this species, harmandi, is in honor of the French Navy surgeon, naturalist, and explorer François-Jules Harmand (1845–1921), who collected the holotype.

4. Discussion

Raiamas guttatus was first described by Day in 1870 as Opsarius guttatus from the Prome to Mandalay reach of Myanmar′s Irrawaddy River [7]. The original description documented essential meristic characters, including dorsal-, pectoral-, pelvic-, and anal-fin ray counts, circumpeduncular scales, and lateral-line scales, alongside some measurements and a brief description of color patterns. A decade later, Sauvage described Bola harmandi based on a unique specimen from Cambodia′s Great Lake (Tonlé Sap Lake, the Mekong River Basin), providing only a brief morphological diagnosis limited to dorsal- and anal-fin ray counts and lateral-line scales, without any measurement data [18]. Subsequent taxonomic revisions synonymized B. harmandi with R. guttatus [15,16,17]. Yang and Hwang described Luciosoma fasciata from seven specimens collected in Xishuangbanna, China (the Mekong River Basin), which was later similarly synonymized as R. guttatus; notably, their description included predorsal scale counts, a feature absent in the prior literature [19]. This study examined 49 specimens of R. guttatus spanning the Irrawaddy, Salween, and Mekong river basins, incorporating the holotype of B. harmandi and 6 syntypes of L. fasciata. Analysis of 25 measurements and 12 meristic characters revealed the Mekong populations possess significantly higher predorsal scale counts (25–28 vs. 21–23 in the other two basins) (Table 3), supporting recognition of R. harmandi as distinct. Consequently, we revalidate R. harmandi with a comprehensive redescription. Critical reassessment confirms L. fasciata (predorsal scales 25–27 for six syntypes) represents a junior synonym of R. harmandi (Table 3). The omission of predorsal scale data in the original descriptions of both species underscores how missing key diagnostic characters can lead to taxonomic errors in morphologically similar congeners.

Examination of live and freshly preserved specimens further revealed interspecific divergence in color patterns. Subadult R. harmandi exhibits slender spots, contrasting with the more rounded spots of R. guttatus. In adults, R. harmandi develops numerous small, irregularly scattered spots, while R. guttatus displays larger spots that merged anteriorly into near-striped formations extending posteriorly (Figure 6c,d). These distinctions align with Day’s description of “two rows of blue spots” [7: 621] versus Yang and Hwang′s description of “irregular black spots anteriorly” in L. fasciata [19: 54]. While this study also observed subtle differences in the distribution of black bands on the caudal fin between live R. harmandi and R. guttatus (Figure 6), this character was excluded from diagnostic features due to limited live material and its poor preservation stability in specimens.

Earlier molecular studies by Tang et al. [10] using RAG1, Rh, Cyt b, and COI genes showed incipient divergence among three R. guttatus specimens but lacked geographic precision to resolve species boundaries. Most prior phylogenetic investigations about Raiamas suffered from inadequate sampling of trans-basin populations, typically representing widespread species with single individuals [8,26,27]. Our study analyzed sequences from nine Raiamas specimens across four countries (including GenBank data from one Laos individual) with explicit drainage representation: four from the Irrawaddy River Basin, one from the Salween River Basin, and four from the Mekong River Basin (Figure 2; Table 1). Results demonstrated the Mekong individuals forming a distinct clade, while the Irrawaddy and Salween specimens clustered in a sister clade (Figure 2). Coupled with the substantial genetic distance between these lineages, our molecular evidence robustly supports morphology-based conclusions: the Mekong populations represent a species distinct from R. guttatus, warranting reinstatement of R. harmandi as valid.

Consistent with previous studies, Raiamas emerges as non-monophyletic [8,9,10,26]. In the present phylogeny, Asian Raiamas species form a monophyletic clade whose sister group comprises an assemblage of multiple Chedrinae lineages, including African Raiamas congeners and endemic African genera: Opsaridium, Leptocypris, Neobola, Rastrineobola, Engraulicypris, and Chelaethiops (Figure 2). This topology demonstrates pronounced evolutionary divergence between Asian and African Raiamas lineages, a pattern previously noted in morphology-based analyses [8]. Current taxonomic frameworks remain complex and unstable due to morphological convergence among Raiamas and allied genera. Future studies integrating molecular systematics, refined morphological comparisons, and osteological examinations will be essential to clarify the classification of this group.

With the revalidation of R. harmandi, the genus Raiamas now comprises 19 valid species, of which 3 are distributed in Asia. Current evidence demonstrates that R. harmandi is widely distributed throughout the Mekong River Basin, with northernmost records extending to Pu′er City (Yunnan Province, China) and southern limits reaching Cambodia, spanning the upper, middle, and lower reaches of the drainage (Figure 1). Although Raiamas have been reported from the Chao Phraya River Basin, Peninsular Malaysia, and Sumatra [20,21,22], the absence of direct specimen examination precludes confirmation of their identity as R. harmandi. This species now represents the easternmost distribution in the Eurasian continent for Raiamas, reflecting a prevailing biogeographic pattern among most Danionidae species. Currently, Opsarius pulchellus and Danio roseus extend eastward to the Red River Basin in the Eurasian continent [51], and only Rasbora steineri extends further eastward to the Pearl River Basin, whereas other danionids are confined eastward to the Mekong River Basin. The reinstatement of R. harmandi highlights potential diversity within trans-basin species and establishes fundamental taxonomic data for investigating danionid diversification and dispersal mechanisms.