Content uploaded by Lin Feng
Author content
All content in this area was uploaded by Lin Feng on Mar 15, 2023
Content may be subject to copyright.
ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Accepted by Y.-H. Zhao: 13 Dec. 2022; published: 5 Jan. 2023 265
Zootaxa 5227 (2): 265–278
https://www.mapress.com/zt/
Copyright © 2023 Magnolia Press Article
https://doi.org/10.11646/zootaxa.5227.2.6
http://zoobank.org/urn:lsid:zoobank.org:pub:9D1A67FB-9ADF-402D-995F-0C9F257FA2CE
Paracanthocobitis putaoensis, a new loach species (Cypriniformes: Nemacheilidae)
from the Irrawaddy basin in northern Myanmar
FENG LIN1,2,3,4#, ZHI-YING CHEN2,3#, KHIN MAR MYINT5 & XIAO-YONG CHEN2,3,4*
1University of Chinese Academy of Sciences, Beijing, 100049, China
�
linfeng@mail.kiz.ac.cn; https://orcid.org/0000-0001-7240-9233
2Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw, 05282, Myanmar
�
chenluchenzhiying@163.com; https://orcid.org/0000-0002-4217-4338
3State Key Laboratory of Genetic Resources and Evolution & Yunnan Key Laboratory of Biodiversity and Ecological
Conservation of Gaoligong Mountain, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan,
650201, China
4Yunnan International Joint Laboratory of Southeast Asia Biodiversity Conservation, Menglun, Yunnan,666303, China
�
chenxy@mail.kiz.ac.cn; https://orcid.org/0000-0002-0924-5560
5Forest Research Institute, Forestry Department, Ministry of Natural Resources and Environment Conservation, Yezin,
Nay Pyi Taw 05282, Myanmar.
�
kmyint.mar@gmail.com; https://orcid.org/0000-0002-4938-9980
#Authors contributed equally to this work
*Corresponding author.
�
chenxy@mail.kiz.ac.cn
Abstract
Paracanthocobitis putaoensis sp. nov. is described based on analysis of morphological and molecular data (cytochrome
c oxidase subunit I—COI). The new species was collected from the Mali Hka River, a tributary of the Irrawaddy River
near Putao in Kachin State in northern Myanmar. It can be easily distinguished from all other species of the genus
Paracanthocobitis by an incomplete lateral line, a suborbital groove in adult males, and a well-developed axillary pelvic
lobe. Phylogenetic relationships of the genus Paracanthocobitis based on the mitochondrial COI locus are revealed for
the first time. Maximum likelihood and Bayesian inference phylogenetic analyses indicate that the new species forms an
independent clade. Both morphological and molecular phylogenetic analyses suggest that P. putaoensis is a new species.
Key words: New loach, Taxonomy, Phylogeny, Mali Hka River
Introduction
Nemacheilidae is a family of cypriniform fishes with more than 760 known species in 49 genera that mostly inhabit
fast-flowing stream environments and is widely distributed in Eurasia (Du et al. 2021; Kottelat 2012a, b; Kottelat
2019; Kottelat & Grego 2020; Zhao et al. 2021). In recent years, many new species within Nemacheilidae have
been described, especially in Southeast Asia and China (e.g., Chen et al. 2020; Dvořák et al. 2022; Jiang et al. 2021;
Kottelat 2022).
Paracanthocobitis was erected as a subgenus of Acanthocobitis Peters 1861 by Grant (2007) with Cobitis
zonalternans as the type species of the subgenus Paracanthocobitis, then was further defined as genus by Singer &
Page (2015). The genus Paracanthocobitis is characterized by the following diagnosis: papilliated pad with medial
notch on each side of lower lip; upper lip with 2–5 rows of papillae and continuous with lower lip; conspicuous
black spot with white outline (an ocellus) at caudal fin base, close to upper extremity of fin base (Kottelat 2012a;
Singer & Page 2015). Twenty species are recognized in genus Paracanthocobitis, and the genus is widely distrib-
uted in South and Southeast Asia, ranging from the Indus basin of Pakistan to the Irrawaddy and Sittang basins in
Myanmar, the Salween drainage in Myanmar and Thailand, the Mekong basin in Cambodia and Laos and the Ma-
lay Peninsula (Arunkumar & Moyon 2019; Kottelat & Vishwanath 2021; Rime et al. 2022; Singer & Page 2015;
Singer et al. 2017). To date, eight members of Paracanthocobitis species, viz., P. adelaideae Singer & Page 2015, P.
LIN ET AL.
266 · Zootaxa 5227 (2) © 2023 Magnolia Press
epimekes Dvořák, Bohlen, Kottelat & Šlechtová 2022, P. linypha Singer & Page 2015, P. mandalayensis (Rendahl
1948), P. nigrolineata Singer, Pfeiffer & Page 2017, P. pictilis (Kottelat 2012a), P. rubidipinnis (Blyth 1860), and
P. zonalternans (Blyth 1860), have been recorded in Myanmar. Among them, six species were distributed in the
Irrawaddy basin.
Three ichthyofaunal diversity surveys in northern Myanmar were conducted from 2014 to 2017, and several
specimens of nemacheilid loaches were collected in tributaries of the Mali Hka River. Further identification con-
firmed that some of these specimens belong to a new species described herein as Paracanthocobitis putaoensis.
Materials and methods
Tissue samples (fin clips) were fixed and stored in 100% ethanol, and the twenty-two associated specimens were
preserved in 10% formalin for long-term storage. Meristic counts and measurements followed Kottelat (1990), Kot-
telat & Freyhof (2007) and Singer & Page (2015), and cephalic sensory system pores counting followed Kottelat
(1990). The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are counted as
“1½”. Measurements were made point to point using digital callipers to 0.1 mm. Whenever possible, counts and
measurements were made on the left side of the specimens. Meristic counts were undertaken using an Olympus
SZ61 microscope. Images of some traits, such as the axillary pelvic lobe, were obtained with an Olympus SZ61 and
a ToupCam microscope digital camera. Specimens were deposited in the repository collections of Southeast Asia
Biodiversity Research Institute (SEABRI), Chinese Academy of Sciences, Nay Pyi Taw, Myanmar, and Kunming
Institute of Zoology (KIZ), Chinese Academy of Sciences, Kunming, Yunnan province, China. Comparative mor-
phological data for unavailable species of Paracanthocobitis were obtained from Kottelat (2012), Singer & Page
(2015), Singer et al. (2017), Arunkumar & Moyon (2019), Kottelat (2021), Rime et al. (2022) and Dvořák et al.
(2022). Abbreviations include ex., examined specimens; SL, standard length; HL, head length; and TL, total length.
The map was developed in QGIS, v3.16.
DNA extraction, PCR and sequencing. Genomic DNA was extracted using a TIAMarine Animals Genome
DNA Kit (Qiagen) following the manufacturer’s instructions. The mitochondrial cytochrome coxidase subunit I
(COI) gene was used as a molecular marker gene and was amplified with the primers FISH_BCL and FISH_BCH
(Singer et al. 2017). Polymerase chain reaction (PCR) was performed in a 30 µL reaction volume containing 15 µL
of 2×Taq PCR MasterMix, 1 µL each of forward and reverse primers (10 μM F2 and R2), 1 µL of template DNA,
and 12 µL of ddH2O. PCR cycling conditions were as follows: initial denaturation at 94°C for 4 min, 35 cycles of
94°C for 30 s, 56°C for 60 s, 72°C for 90 s, and 10 min at 72°C. Unpurified PCR products were sent to TianGen
Biotech (Kunming) Co., Ltd. for bidirectional Sanger sequencing.
Molecular data analysis. Phylogenetic analysis was performed based on 51 sequences, including 10 newly
generated COI barcodes and 41 sequences (36 already published COI sequences of Paracanthocobitis species and
5 other nemacheilid loaches as outgroups) obtained from GenBank (accession numbers in Table 1). Sequences were
assembled in the SeqMan II module of the DNASTAR Lasergene software package (Burland 2000) and aligned
by using the Clustal W algorithm implemented in MAGE X (Kumar et al. 2018), and then checked manually for
inconsistencies. The aligned sequence matrix of the COI gene was 603 bp, which was used for subsequent analysis.
Maximum likelihood (ML) phylogenies were inferred in IQ-TREE (Nguyen et al. 2015) with ultrafast bootstrap
support (Minh et al. 2013) for 1000 iterations. Phylogenetic relationships were estimated with Bayesian infer-
ence (BI) using MrBayes v3.2.6 (Ronquist et al. 2012) in PhyloSuite v1.2.2 (Zhang et al. 2020). The best-fitting
nucleotide substitution models as determined by ModelFinder (Kalyaanamoorthy et al. 2017) based on the Bayesian
Information Criterion (BIC) for ML and BI phylogenies were TPM2+I+G4 and HYK+I+G4, respectively. The BI
analysis was performed by two independent runs of Markov Chain Monte Carlo (MCMC), each with one cold chain
and three hot chains, for 10 × 106 generations (sampling frequency of each 1000 generations), and the first 25% trees
were discarded as burn-in. Convergence was inferred when the average standard deviation of split frequencies fell
below 0.01. The phylogenetic tree was edited in FigTree v1.4.4.
PARACANTHOCOBITIS PUTAOENSIS Zootaxa 5227 (2) © 2023 Magnolia Press · 267
TABLE 1. Information on samples and sequences used for phylogenetic analysis
GenBank
Accession
No.
Species Voucher No. Country Source
MK572429 Paracanthocobitis abutwebi NRM 67004 Bangladesh Rahman et al., 2019
MK572434 Paracanthocobitis abutwebi NRM 68228 Bangladesh
MK572435 Paracanthocobitis abutwebi NRM 67929 Bangladesh
KX576654 Paracanthocobitis adelaideae PA 3001 India Barman et al., 2018
MK480382 Paracanthocobitis adelaideae NMHS FES41 India Kundu et al., 2019
MK480383 Paracanthocobitis adelaideae NMHS FES42 India
KT005602 Paracanthocobitis aurea WILD-14-PIS-115 India GenBank/Unpublished
KT005603 Paracanthocobitis aurea WILD-14-PIS-117 India
FJ459449 Paracanthocobitis botia NBFGR: AP8068A India Lakra et al., 2016
FJ459450 Paracanthocobitis botia NBFGR: AP8068B India
FJ459451 Paracanthocobitis botia NBFGR: AP8068C India
KY290076 Paracanthocobitis botia ZSI F273 India Lasker et al.,2018
OP723456 Paracanthocobitis linypha S 20182668 Myanmar This study
OP723455 Paracanthocobitis linypha S 20192258 Myanmar
MK572438 Paracanthocobitis mackenziei DU 6221 Bangladesh Rahman et al., 2019
MK572439 Paracanthocobitis mackenziei NRM 67078 Bangladesh
MF289059 Paracanthocobitis maekhlongensis UF 182864 Thailand Singer et al., 2017
MF289060 Paracanthocobitis maekhlongensis NFI 4853 Thailand
MF289055 Paracanthocobitis mandalayensis UF 181110 Thailand
MF289056 Paracanthocobitis mandalayensis UF 181110 Thailand
OP723458 Paracanthocobitis mandalayensis SS 20192332 Myanmar This study
OP723457 Paracanthocobitis mandalayensis SS 20192333 Myanmar
KT005593 Paracanthocobitis mooreh BNHS FWF 49 India Keskar et al., 2018
KT005594 Paracanthocobitis mooreh WILD-13-PIS-050 India
KX946753 Paracanthocobitis mooreh KF 150 India Patil et al., 2018
KX946754 Paracanthocobitis mooreh KF 163 India
MF289052 Paracanthocobitis nigrolineata UF 176411 Thailand Singer et al., 2017
MF289053 Paracanthocobitis nigrolineata UF 181149 Thailand
MF289067 Paracanthocobitis nigrolineata UF 188055 Thailand
MF289068 Paracanthocobitis nigrolineata UF 188055 Thailand
MF289057 Paracanthocobitis phuketensis UF 182833 Thailand
MF289061 Paracanthocobitis phuketensis UF 230643 Thailand
MF289062 Paracanthocobitis phuketensis UF 236051 Thailand
MF289049 Paracanthocobitis pictilis UF 172927 Thailand
OP723460 Paracanthocobitis putaoensis QT 20170096 Myanmar This study
OP723461 Paracanthocobitis putaoensis QT 20170116 Myanmar
OP723462 Paracanthocobitis putaoensis QT 20170117 Myanmar
OP723463 Paracanthocobitis putaoensis QT 20170132 Myanmar
OP723464 Paracanthocobitis putaoensis QT 20170133 Myanmar
OP723459 Paracanthocobitis rubidipinnis CXY 20170103 Myanmar
......continued on the next page
LIN ET AL.
268 · Zootaxa 5227 (2) © 2023 Magnolia Press
TABLE 1. (Continued)
GenBank
Accession
No.
Species Voucher No. Country Source
AP012137 Paracanthocobitis urophthalma Unkonwn Sri Lanka GenBank/ Unpublished
MF289069 Paracanthocobitis zonalternans UF 188197 Thailand Singer et al., 2017
MF289070 Paracanthocobitis zonalternans UF 188197 Thailand
MF289071 Paracanthocobitis zonalternans UF 188198 Thailand
MF289072 Paracanthocobitis zonalternans UF 188198 Thailand
MF289073 Paracanthocobitis zonalternans UF 188197 Thailand
Outgroup
KU928267 Acanthocobitis pavonacea ZFMK-TIS-2567411 India Freyhof et al., 2016
KX384765 Indoreonectes evezardi BNHS FWF 300 India Keskar et al., 2018
MG018976 Indoreonectes keralensis WILD-17-PIS-343 India
MF289074 Nemacheilus masyae UF 176446 Thailand Singer et al., 2017
JN177233 Schistura fasciolata IHB 0706004 China Liu et al., 2012
Paracanthocobitis putaoensis sp. nov., Lin, Chen & Chen
(Figs 1–4)
Holotype. KIZ 2015006395 (SEABRI 20151117), 55.7 mm standard length (SL), male; Tanjar Stream, passing
Lone Shar Yan Village, Putao District, Kachin State, Myanmar (approx. 27°08'18.92"N, 97°33'34.38"E; ~422 m
a.s.l.); col. Xiao-Yong Chen, Tao Qin, Shu-Sen Shu, and Yunn Mi Mi Kyaw, 26 December 2015.
Paratypes. SEABRI 20140216–17, 19, 21–22, 25, 27–29, 9 ex., 42.5–74.5 mm SL, tributary of Mali Hka River
in NaungMun, Putao District, Kachin State, Myanmar (approx. 27°30'15.3"N, 97°48'48.2"E; ~543 m a.s.l.), col.
Tao Qin; 30 November 2014 to 7 December 2014. SEABRI 20151113, 20151116, 20151118, 3 ex., 36.6–66.0 mm
SL, same data as holotype. KIZ 2015006396–98 (SEABRI 20151247–49), 3 ex., 41.2–72.3 mm SL, Chan Khaung
Stream, Putao District, Kachin State, Myanmar (approx. 27°24'32.09"N, 97°18'57.05"E; ~405 m a.s.l.), col. Xiao-
Yong Chen, Tao Qin, Shu-Sen Shu, and Yunn Mi Mi Kyaw; 28 December 2015.
Additional material. SEABRI 20151114, 1 ex., 55.1 mm SL, same data as holotype, used for physical dissection.
Material used in molecular genetic analysis. SEABRI-QT 20170096, 1 ex., 62.8 mm SL, Putao District: a
stream of Mali Hka River, 27°40'20.69"N, 97°23'2.70"E (GenBank accession numbers: OP723460).—SEABRI-
QT 20170116-117, 132-133, 4 ex., 54.3–66.3 mm SL, Putao District: a stream of Mali Hka River, 27°41'57.54"N,
97°24'7.29"E (GenBank accession numbers: OP723461-OP723464).
Description: General appearance is shown in Figures 1–2. Morphometric data for the holotype and 15 para-
types are shown in Table 2. Body moderately elongated and robust, largest known specimen 74.5 mm SL. Body
cylindrical anteriorly (between pectoral-fin and dorsal-fin origin) and slightly compressed posteriorly, especially
caudal peduncle. Dorsal profile continuous with no hump between head and body, except the inconspicuous slight
depression on occipital margin. Deepest and widest point of body at or just anterior to dorsal-fin origin; body depth
slowly increasing to dorsal-fin origin and then appreciably decreasing to end of dorsal-fin base, postdorsal body
depth almost uniform to caudal-fin base. Ventral profile almost flat to anal-fin origin, then slightly rising to caudal-
Diagnosis: Paracanthocobitis putaoensis can be readily distinguished from all other species of Paracanthoco-
bitis by the combination of horizontally oriented suborbital groove in males; incomplete lateral line, with 39–54
pores, extending to anus but not surpassing origin of anal-fin; axillary pelvic lobe present; 9½–10½ (usually 10½)
branched dorsal-fin rays; 11–12 (usually 11) pectoral-fin rays; and caudal fin with 3–5 dark bands.
fin base.
PARACANTHOCOBITIS PUTAOENSIS Zootaxa 5227 (2) © 2023 Magnolia Press · 269
FIGURE 1. Paracanthocobitis putaoensis sp. nov. after fixation. KIZ 2015006395, holotype, male, 55.7 mm SL. a. lateral
view; b. dorsal view; c. ventral view.
FIGURE 2. Paracanthocobitis putaoensis sp. nov. after fixation, paratypes; a. SEABRI 20140229, 65.5 mm SL; b. SEABRI
20151113, 42.3 mm SL; and c. SEABRI 20151249, 41.2 mm SL.
LIN ET AL.
270 · Zootaxa 5227 (2) © 2023 Magnolia Press
TABLE 2. Morphological measurements of Paracanthocobitis putaoensis, holotype and 15 paratypes. Ranges and means
include values of holotype.
Holotype Holotype & Paratypes
mean min max SD
Total length (mm) 66.8 70.2 45.8 88.3 13.3
Standard length (mm) 55.7 57.8 36.6 74.5 11.2
In percent of standard length
Dorsal head length 20.6 20.4 18.7 23.1 1.2
Lateral head length 22.6 23.4 21.0 28.2 1.6
Predorsal length 48.5 47.2 43.5 49.8 1.8
Prepelvic length 52.4 52.7 50.3 54.9 1.2
Preanal length 79.5 79.9 76.8 82.6 1.6
Preanus length 74.5 75.0 70.7 77.2 1.8
Snout length 9.7 9.4 7.4 10.7 0.8
Body depth 18.1 17.5 14.8 20.5 2.0
Length of caudal peduncle 14.9 13.9 12.0 16.8 1.1
Depth of caudal peduncle 14.0 11.9 10.9 14.0 0.9
Head depth (at eye) 11.1 11.4 10.3 12.8 0.8
Head depth (at nape) 13.1 13.1 11.6 15.0 1.1
Body width (dorsal origin) 14.4 12.1 10.4 15.5 1.8
Body width (anal origin) 7.5 6.8 5.5 8.8 1.2
Head width (at nares) 9.9 8.3 7.1 10.0 1.0
Maximum head width 14.7 14.9 12.1 17.0 1.3
Eye diameter 3.9 4.0 3.2 6.6 0.9
Interorbital width 5.7 6.9 4.7 10.1 1.1
Dorsal-fin length 18.3 19.0 17.2 21.9 1.2
Pectoral-fin length 18.0 17.6 14.9 19.8 1.5
Pelvic-fin length 14.9 15.0 13.4 16.5 0.7
Anal-fin length 16.3 15.9 13.8 17.2 0.9
Length of upper lobe of caudal fin 19.4 21.1 19.4 24.0 1.4
Length of lower lobe of caudal fin 18.1 20.8 18.1 23.2 1.2
Length of median ray of caudal fin 18.1 20.0 18.1 21.9 1.0
In percent of lateral head length
Snout length 42.9 40.3 30.7 45.0 3.8
Head width 65.1 63.8 49.0 72.1 6.1
Head depth (at eye) 49.2 48.7 43.1 53.9 3.4
Head depth (at nape) 57.9 56.0 49.1 62.5 4.3
Eye diameter 17.5 17.2 14.7 26.9 3.1
Interorbital width 25.4 29.6 19.2 42.0 5.0
Pectoral-fin length 79.4 75.5 62.9 89.8 7.4
Pelvic-fin length 65.9 64.1 51.7 72.7 4.7
Anal-fin length 72.2 68.2 57.8 78.1 5.9
Head moderately depressed and triangular when viewed dorsally, snout obtusely rounded. In lateral view,
cheeks slightly swollen, snout slightly pointed. Anterior nostril pierced in front of a flap-like tube; posterior nostril
adjacent to anterior nostril. Eye relatively small, eye diameter 14.7–26.9% HL. Mouth inferior, moderately large
and strongly arched, gape about twice as wide as long. Lips thick and fleshy with well-developed papillae; upper lip
with 3–4 rows of papillae, contiguous with lower lip, and a small median incision on upper lip; large thick pad with
PARACANTHOCOBITIS PUTAOENSIS Zootaxa 5227 (2) © 2023 Magnolia Press · 271
dense bulbous papillae on sides of lower lip; pads separated by a narrow median interruption (Fig. 3 a). Processus
dentiformis on upper jaw present, and no median notch on lower jaw. Three pairs of barbels; maxillary barbels lon-
gest, reaching vertical of posterior rim of eye; inner rostral barbels reaching anterior margin of nostril, outer rostral
barbels almost reaching vertical of anterior margin of orbit.
Body entirely covered with scales. Dorsal-fin origin anterior to pelvic-fin origin, and slightly closer to snout
tip than to caudal-fin base; dorsal-fin with 4 simple and 9½ (6) or 10½ (10) branched rays, the 1st or 2nd branched
ray longest; posterior margin of dorsal-fin truncate and distal margin above vertical of anus. Pectoral-fin margin
slightly convex, with 1 simple and 10 (13) or 11 (3) branched rays; tip of adpressed fin reaching about 1/2 to 3/5
distance from pectoral-fin origin to pelvic-fin origin. Pelvic-fin origin at vertical line of third branched ray of dorsal
fin, and slightly closer to pectoral-fin origin than to anal-fin origin or at midpoint of them. Pelvic fin with 1 simple
and 7 branched rays, and tip of adpressed fin not reaching anus. Axillary pelvic lobe present (Fig. 3 b). Anus closer
to anal-fin origin than to pelvic-fin tip. Anal-fin origin closer to caudal-fin base than to pelvic-fin origin. Anal fin
small with convex margin, with 3 simple and 5½ branched rays, not reaching caudal-fin base. Caudal fin slightly
emarginate, with 8+8 branched rays. Caudal-peduncle length 1.1–1.2 times its depth.
Lateral line incomplete, with 39–54 pores (6), extending at vicinity of anus and not surpassing anal-fin inser-
tion. Cephalic sensory system (6) with 4+9–11 infraorbital pores, 7–8 supraorbital pores, 3 supratemporal pores,
and 8 preoperculo-mandibular pores. Intestine with a loop behind stomach, extending forward to anterior extremity
of stomach (Fig. 3 c).
FIGURE 3. Morphological characteristics of Paracanthocobitis putaoensisis; a. lower lip (KIZ 2015006395, holotype, male,
55.7 mm SL); b. lateral view of the axillary pelvic lobe (SEABRI 20151118, paratype, 66.0 mm SL); c. ventral view of the
digestive tract (SEABRI 20151114); d. lateral view of the suborbital groove in males (SEABRI 20151113, paratype, male, 42.3
mm SL).
LIN ET AL.
272 · Zootaxa 5227 (2) © 2023 Magnolia Press
Colouration: In preservative, yellowish brown on background of head and body, whitish on belly; head with
2–3 brown transverse blotches, snout, and cheeks with many small dark spots. Body with 3 rows of large blotches
and 1 row of small, irregular paler spots close to ventral side. The 1st row is 10–15 brown saddles across dorsum
(3–4 predorsal, 3–4 subdorsal and 4–7 postdorsal). The 3rd row composed of 8–10 large brown irregular blotches
centered along lateral line and extending to ventral side, some becoming slightly slanted, shape of blotches variable
from elongated oval, oval, heart-shaped to triangular; 2nd row composed of irregular different-sized blotches or spots
inserted above lateral line at interspace between 1st and 3rd rows (saddles and the blotches along lateral line). Black
ocellus close to dorsal margin of caudal peduncle. Dorsal fin hyaline, with 3–4 longitudinal rows of dark brown
spots. Caudal fin with 3–5 (usually 4) almost vertical rows of dark bands on rays, other fins hyaline.
In life (Fig. 4), body and head light brown; belly paler; 2–3 dark brown transverse blotches on head; many dense
and diverse dark brown blotches on body; subrotund black ocellus on upper 1/3 of caudal peduncle; pectoral, anal,
pelvic, and caudal fins pale yellow; several light black spots on pectoral-fin and caudal-fin rays.
FIGURE 4. Live individual of Paracanthocobitis putaoensis, from a small stream on the way from Zeyar Dan to Hponkanrazi
on Dec., 15, 2015. Photo by Chen Xiao-Yong.
Sexual dimorphism: Adult males distinguished by presence of well-defined suborbital groove, extending from
lower rim of eye to upper base of inner rostral barbel (Fig. 3 d). Females without suborbital groove or with a shal-
lower, less defined groove in similar position.
Distribution and habitat: Paracanthocobitis putaoensis is known from the type locality, Tanjar Stream, Chan
Khaung Stream and a tributary of the Mali Hka River in Naung Mun, all of which are tributaries of the Mali Hka
River, Myanmar (Fig. 5). This species inhabits slow flowing streams with a substrate of small gravel and stones (Fig.
6). Other fishes collected with P. putaoensis include Psilorhynchidae: Psilorhynchus brachyrhynchus; Cyprinidae:
Danio flagrans, Tor yingjiangensis, Neolissochilus compressus, Opsarius barinoides, Poropuntius burtoni, Pethia
tiantian, Pethia thelys, Garra qiaojiensis and Garra bispinosa; Nemacheilidae: Schistura sikmaiensis; Amblycepi-
tidae: Amblyceps murraystuarti; Bagridae: Batasio procerus; Mastacembelidae: Mastacembelus armatus; Badidae:
Badis pyema; Channidae: Channa burmanica.
Etymology: The specific name “putaoensis” refers to Putao District, Kachin State, Myanmar.
Discussion
Singer & Page (2015) proposed a hypothesis that the genus Paracanthocobitis can be divided into three species
complexes based on morphological characteristics (a suborbital flap or groove in the male, more than or less than
10½ dorsal-fin rays, and body size), viz. the P. botia, P. zonalternans, and P. mandalayensis complexes. A suborbital
flap is a shared feature of members of the P. botia and P. zonalternans complexes, whereas species of the P. zonal-
ternans complex have an incomplete lateral line (vs. complete lateral line in P. botia complex, except in P. mooreh
and P. linypha), and species of the P. mandalayensis complex have a suborbital groove and a complete lateral line
(Singer et al. 2017; Rime et al. 2022).
PARACANTHOCOBITIS PUTAOENSIS Zootaxa 5227 (2) © 2023 Magnolia Press · 273
FIGURE 5. Distribution map of Paracanthocobitis putaoensis; star for locality of holotype, triangle for those of paratypes,
circle for locality of specimens used in molecular genetic analysis.
LIN ET AL.
274 · Zootaxa 5227 (2) © 2023 Magnolia Press
FIGURE 6. Habitat of Paracanthocobitis putaoensis; Myanmar: Chan Khaung Stream, Dec. 28, 2015.
Paracanthocobitis putaoensis has a suborbital groove in the male and can be provisionally placed in the P. man-
dalayensis complex, but we speculate that it should be recognized as an independent group, due to its unique com-
bination of morphological characteristics: lateral line incomplete, reaching at most to anal-fin insertion, a suborbital
groove, axillary pelvic lobe present, 9½–10½ branched dorsal-fin rays and large body size (most adult individuals
more than 50 mm SL).
Paracanthocobitis putaoensis can be further differentiated from the P. mandalayensis species complex (includ-
ing P. canicula, P. maekhlongensis, P. mandalayensis, and P. pictilis) by having an incomplete (vs. complete) lateral
line. It is further distinguished from P. canicula in having 9½–10½ (vs. 11½–12½) branched dorsal-fin rays, 11–12
(usually 11, vs. 13) pectoral-fin rays, and 3–5 (vs. 5–7) dark bands on caudal fin (Singer & Page 2015); from P.
maekhlongensis in having 9½–10½ (vs. 12½–13½) branched dorsal-fin rays, 11–12 (usually 11) vs. 12–14 (usually
13) pectoral-fin rays, 8 vs. 7–8 (usually 7) pelvic-fin rays, and 3–5 (vs.7–8) dark bands on caudal fin (Singer & Page
2015); from P. mandalayensis in having 9½–10½ (usually 10½) vs. 10½–11½ (usually 11½) branched dorsal-fin
rays, 11–12 (usually 11) vs. 12–13 (usually 13) pectoral-fin rays, 3–5 (vs. 7–11) dark bands on caudal fin (Singer &
Page 2015); from P. pictilis in having 9½–10½ (usually 10½) vs. 12½–13½ (usually 12½) branched dorsal-fin rays,
11–12 (usually 11) vs. 13–15 (usually 13) pectoral-fin rays, 3–5 (vs. 6–8) dark bands on caudal fin.
Paracanthocobitis putaoensis differs from the P. botia species complex (including P. abutwebi, P. adelaideae,
P. aurea, P. botia, P. hijumensis, P. linypha, P. mackenziei, P. mooreh, P. rubidipinnis, and P. urophthalma) by having
a suborbital groove (vs. a suborbital flap) in males, and an incomplete (vs. complete) lateral line except P. linypha,
P. mooreh, and P. urophthalma. It is further distinguished from P. linypha, P. mooreh, and P. urophthalma by pres-
ence of an axillary pelvic lobe (vs. absence), and 39–54 lateral line pores (vs. 22–42 in P. linypha, vs. 28–39 in P.
mooreh, and 59–71 in P. urophthalma, respectively); from P. linypha in having large irregular blotches along and
below the lateral line (vs. thin bars on side of body); from P. mooreh in having 11–12 (usually 11) vs. 11–13 (usu-
ally 12) pectoral-fin ray, and a longer head (21.0–28.2% SL vs. 19.6–22.3% SL); from P. urophthalma in having
9½–10½ (usually 10½) vs. 10½–12½ (usually 11½) branched dorsal-fin rays, and irregular blotches on side of body
(vs. large bars extending form dorsum to venter) (Singer & Page 2015).
PARACANTHOCOBITIS PUTAOENSIS Zootaxa 5227 (2) © 2023 Magnolia Press · 275
Paracanthocobitis putaoensis is distinguished from six species in the P. zonalternans complex (including P.
epimekes, P. marmorata, P. nigrolineata, P. triangula, P. phuketensis and P. zonalternans) by having a suborbital
groove (vs. a suborbital flap) in males, 8 (vs. 6–7) pelvic-fin rays, and a larger adult size (more than 50 mm SL vs.
less than 50 mm SL) (Singer et al. 2017; Dvořák et al. 2022). It can be further distinguished from the P. zonalternans
complex by an incomplete lateral line, extending to vicinity of anus and not surpassing origin of anal fin (vs. usually
ending close to dorsal-fin insertion).
Based on the tree topology resulting from maximum likelihood and Bayesian inference reconstruction of the
COI barcodes for 21 nemacheilid loaches (Fig. 7), Paracanthocobitis putaoensis was supported as a new species.
Meanwhile, we found that the genus Paracanthocobitis formed a monophyletic assemblage and was estimated to be
sister to Acanthocobitis and Indoreonectes. Our results further supported molecular data analysis in Šlechtová et al.
(2007) and Freyhof et al. (2016), although their research did not focus on Paracanthocobitis. The validity of Para-
canthocobitis species (except P. canicula, P. epimekes, P. hijumensis, P. marmorata, and P. triangula) were further
established. In addition, the ML and BI phylogenies consistently showed that genus Paracanthocobitis was divided
into two major clades and three groups (group 1: P. abutwebi, P. adelaideae, P. aurea, P. botia, P. mackenziei, P.
mooreh, and P. urophthalma; group 2: P. linypha, P. maekhlongensis, P. mandalayensis, P. pictilis, P. rubidipinnis,
P. nigrolineata, P. phuketensis, and P. zonalternans), and P. putaoensis formed an independent branch (group 3)
with good support. Therefore, the molecular phylogeny of this study confirmed that Paracanthocobitis botia, P.
zonalternans, and P. mandalayensis complexes formed separate clades, which conforms to Singer & Page’s (2015)
morphological conjecture, except that P. linypha and P. rubidipinnis were not nested within the P. botia complex. A
lot of studies showed that the evolutionary tree constructed by one mitochondrial marker is not completely consis-
tent with the species tree, which may be caused by incomplete lineage sorting or gene introgression (Doyle, 1997;
Szllosiet al. 2013; Zhou et al. 2017). The molecular phylogeny also supported our hypothesis that P. putaoensis
forms an independent clade and may be a key unit in the morphological evolution of Paracanthocobitis. Given that
we studied only one DNA gene marker (COI), we did not conduct an in-depth analysis of ancestral character state
reconstruction. Molecular data of P. mandalayensis in the Wang River drainage and Irrawaddy River drainage did
not form a monophyletic group, indicating that it may have two separate evolutionary origins or be a hidden species.
A systematic review and phylogeny based on more molecular markers of P. mandalayensis is needed to evaluate its
taxonomic status.
FIGURE 7. Phylogenetic relationships of Paracanthocobitis species based on COI gene sequences reconstructed by Bayesian
inference (BI); values above and below branches correspond to BI posterior probabilities and maximum-likelihood (ML) boot-
strap support, respectively.
LIN ET AL.
276 · Zootaxa 5227 (2) © 2023 Magnolia Press
Comparative material
Paracanthocobitis linypha: SEABRI uncat, 34 ex.; 37.2–44.4 mm SL, Manipur River, Kalemyo District, Chin State,
Myanmar; SEABRI uncat, 36 ex; 25.6–39.1 mm SL, Irrawaddy, Myanmar; SEABRI-SS 20182668, 1 ex, 48.4
mm SL, Nao Kuang stream, a stream of Indawgyi Lake, Myanmar; SEABRI-SS 20192258, 1 ex, 39.4 mm SL,
Lae Pon stream, a stream of Indawgyi Lake, Myanmar.
Paracanthocobitis mandalayensis: SEABRI-CXY 20150799–20150805, 7 ex.; 30.7–56.7 mm SL, Nan Shae Stream,
Putao District, Kachin State, Myanmar; SEABRI-SS 20192332–33, 2 ex, 63.9–67 mm SL, Nyaung Bin Thar
stream, a stream of Indawgyi Lake, Myanmar.
Paracanthocobitis nigrolineata: SEABRI-CXY 20160199, 20160212–20160213, 3 ex., 27.8–31.9 mm SL, a tribu-
tary of Myitnge River, Putao District, Kachin State, Myanmar.
Paracanthocobitis rubidipinnis: SEABRI-CXY 20170103, 1 ex., 64.1 mm SL, fish market of Sagaing, Myanmar.
Acknowledgements
We would like to express our sincere gratitude to the Ministry of Natural Resources and Environmental Protection
of Myanmar for permission to conduct field work. We sincerely thank Tao Qin and Shu-Sen Shu (Kunming Institute
of Zoology, Chinese Academy of Sciences) and Yunn Mi Mi Kyaw (Forest Research Institute, Myanmar) for their
assistance in the field. We also thank Rui-Chang Quan and Ren Li (Xishuangbanna Tropical Botanical Garden,
Chinese Academy of Sciences) for their field support. We thank Meng-Fang Chen (Anhui Normal University) for
her assistance in molecular experiments, Khin Yadanar Htay (Southeast Asia Biodiversity Research Institute) for
helping to check samples and Dr. Christine Watts for helping to polish the manuscript. This work was funded by
the National Natural Science Foundation of China (Grant No. 31872202), the Southeast Asia Biodiversity Research
Institute, Chinese Academy of Sciences (Y4ZK111B01), and the Yunnan Province Science and Technology Depart-
ment (202203AP140007).
References
Arunkumar, L. & Moyon, W.A. (2019) Paracanthocobitis tumitensis, a new species of zipper loach from Manipur, north-eastern
India (Cypriniformes: Nemacheilidae). Species, 20, 101–109.
Barman, A.S., Singh, M., Singh, S.K., Saha, H., Singh, Y.J., Laishram, M. & Pandey, P.K. (2018) DNA Barcoding of Freshwater
Fishes of Indo-Myanmar Biodiversity Hotspot. Scientific Reports, 8, 8579.
https://doi.org/10.1038/s41598-018-26976-3
Burland, T.G. (2000) DNASTAR’s Lasergene sequence analysis software. Methods in Molecular Biology, 132, 71–91.
https://doi.org/10.1385/1-59259-192-2:71
Chen, M.F., Myint, K.M., Chu, L. & Chen, X.Y. (2020) Two new species of loaches from the Irrawaddy River basin, Chin State,
Myanmar (Teleostei: Cypriniformes: Nemacheilidae). Zootaxa, 4895 (1), 86–102.
https://doi.org/10.11646/zootaxa.4895.1.4
Doyle, J.J. (1997) Trees within trees: genes and species, molecules and morphology. Systematic biology, 46 (3), 537–553.
https://doi.org/10.1093/sysbio/46.3.537
Du, L.N., Yang, J., Min, R., Chen, X.Y. & Yang, J.X. (2021) A review of the Cypriniform tribe Yunnanilini prokofiev, 2010 from
China, with an emphasis on five genera based on morphologies and complete mitochondrial genomes of some species.
Zoological Research, 42 (3), 310–334.
https://doi.org/10.24272/j.issn.2095-8137.2020.229
Dvořák, T., Bohlen, J., Kottelat, M. & Šlechtová, V. (2022) Paracanthocobitis epimekes, a new loach species from Myanmar
and Thailand (Teleostei: Nemacheilidae). Ichthyological Exploration of Freshwaters, 1174, 1–11.
Freyhof, J., Geiger, M.F., Golzarianpour, K. & Patimar, R. (2016) Sasanidus, a new generic name for Noemacheilus kerman-
shahensis Bănărescu & Nalbant, with discussion of Ilamnemacheilus and Schistura (Teleostei; Nemacheilidae). Zootaxa,
4107 (1), 65–80.
https://doi.org/10.11646/zootaxa.4107.1.3
Grant, S. (2007) A new subgenus of Acanthocobitis Peters, 1861 (Teleostei: Nemacheilidae). Ichthyofile, 2, 1–9.
Jiang, W.S., Zhao, Y.P., Du, L.N. & Wang, M. (2021) Yunnanilus chuanheensis, a new loach species (Cypriniformes: Nemachei-
lidae) from the upper Lixianjiang River in Yunnan, China. Zoological Research, 42 (2), 241–245.
https://doi.org/10.24272/j.issn.2095-8137.2020.287
PARACANTHOCOBITIS PUTAOENSIS Zootaxa 5227 (2) © 2023 Magnolia Press · 277
Kalyaanamoorthy, S., Minh, B.Q., Wong, T., von Haeseler, A. & Jermiin, L.S. (2017) ModelFinder: fast model selection for
accurate phylogenetic estimates. Nature methods, 14 (6), 587–589.
https://doi.org/10.1038/nmeth.4285
Keskar, A., Raghavan, R., Paingankar, M.S., Kumkar, P. & Dahanukar, N. (2018) Molecular phylogeny unveils hidden diversity
of hillstream loaches (Cypriniformes: Cobitoidea) in the northern Western Ghats of India. Meta Gene, 17, 237–248.
https://doi.org/10.1016/j.mgene.2018.07.002
Kottelat, M. (1990) Indochinese nemacheilines. A revision of nemacheiline loaches (Pisces: Cypriniformes) of Thailand, Bur-
ma, Laos, Cambodia and southern Viet Nam. Verlag Dr. Friedrich Pfeil, München, 262 pp.
Kottelat, M. (2012a) Acanthocobitis pictilis, a new species of loach from Myanmar and Thailand (Teleostei: Nemacheilidae).
Zootaxa, 3327 (1), 45–52.
https://doi.org/10.11646/zootaxa.3327.1.4
Kottelat, M. (2012b) Conspectus cobitidum: an inventory of the loaches of the world (Teleostei: Cypriniformes: Cobitoidei).
Raffles Bulletin of Zoology, Supplement 26, 1−199.
Kottelat, M. (2019) Rhyacoschistura larreci, a new genus and species of loach from Laos and redescription of R. suber (Tele-
ostei: Nemacheilidae). Zootaxa, 4612 (2), 151–170.
https://doi.org/10.11646/zootaxa.4612.2.1
Kottelat, M. (2022) Nemacheilus pezidion, a new species of loach from southern Laos (Teleostei: Nemacheilidae). Zootaxa,
5129 (1), 92–104.
https://doi.org/10.11646/zootaxa.5129.1.5
Kottelat, M. & Freyhof, J. (2007) Handbook of European freshwater fishes. Kottelat, Cornol & Freyhof, Berlin, xiv + 646 pp.
Kottelat, M. & Grego, J. (2020) Kayahschistura lokalayensis, a new genus and species of cave fish from Myanmar (Teleostei:
Nemacheilidae). The Raffles Bulletin of Zoology, Supplement 35, 179–185.
Kottelat, M. & Vishwanath, W. (2021) Type locality and synonymy of Paracanthocobitis marmorata and notes on Acanthoco-
bitis (Teleostei: Nemacheilidae). Raffles Bulletin of Zoology, 69, 13–18.
https://doi.org/10.26107/RBZ-2021-0003
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. (2018) MEGA X: molecular evolutionary genetics analysis across com-
puting platforms. Molecular Biology and Evolution, 35 (6), 1547−1549.
https://doi.org/10.1093/molbev/msy096
Kundu, S., Chandra, K., Tyagi, K., Pakrashi, A. & Kumar, V. (2019) DNA barcoding of freshwater fishes from Brahmaputra
River in Eastern Himalaya biodiversity hotspot. Mitochondrial DNA B Resources, 4 (2), 2411–2419.
https://doi.org/10.1080/23802359.2019.1637290
Lakra, W.S., Singh, M., Goswami, M., Gopalakrishnan, A., Lal, K.K., Mohindra, V., Sarkar, U.K, Punia, P.P., Singh, K.V., Bhatt,
J.P. & Ayyappan, S. (2016) DNA barcoding Indian freshwater fishes. Mitochondrial DNA. Part A, DNA Mapping, Sequenc-
ing, and Analysis, 27 (6), 4510–4517.
https://doi.org/10.3109/19401736.2015.1101540
Laskar, B.A., Kumar, V., Kundu, S., Darshan, A., Tyagi, K. & Chandra, K. (2018) DNA barcoding of fishes from River Diphlu
within Kaziranga National Park in northeast India. Mitochondrial DNA Part A, DNA Mapping, Sequencing, and Analysis,
30 (1), 126–134.
https://doi.org/10.1080/24701394.2018.1463373
Liu, S.Q., Mayden, R.L., Zhang, J.B., Yu, D., Tang, Q.Y., Deng, X. & Liu, H.Z. (2012) Phylogenetic relationships of the Co-
bitoidea (Teleostei: Cypriniformes) inferred from mitochondrial and nuclear genes with analyses of gene evolution. Gene,
508 (1), 60–72.
https://doi.org/10.1016/j.gene.2012.07.040
Minh, B.Q., Nguyen, M.A. & von Haeseler, A. (2013) Ultrafast approximation for phylogenetic bootstrap. Molecular biology
and evolution, 30 (5), 1188–1195.
https://doi.org/10.1093/molbev/mst024
Nguyen, L.T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015) IQ-TREE: a fast and effective stochastic algorithm for
estimating maximum-likelihood phylogenies. Molecular biology and evolution, 32 (1), 268–274.
https://doi.org/10.1093/molbev/msu300
Patil, T.S., Jamdade, R.A., Patil, S.M., Govindwar, S.P, & Muley, D.V. (2018) DNA barcode based delineation of freshwater
fishes from northern Western Ghats of India, one of the world's biodiversity hotspots. Biodiversity and Conservation, 27,
3349–3371.
https://doi.org/10.1007/s10531-018-1604-0
Rahman, M.M., Norén, M., Mollah, A.R. & Kullander, S.O. (2019) Building a DNA barcode library for the freshwater fishes of
Bangladesh. Scientific Reports, 9, 9382.
https://doi.org/10.1038/s41598-019-45379-6
Rime, G., Tamang, L. & Das, D.N. (2022) Paracanthocobitis hijumensis, a new species of zipper loach from the Arunachal
Himalaya, northeastern India (Cypriniformes: Nemacheilidae). Zootaxa, 5115 (1), 122–130.
https://doi.org/10.11646/zootaxa.5115.1.8
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsen-
beck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space.
LIN ET AL.
278 · Zootaxa 5227 (2) © 2023 Magnolia Press
Systematic biology, 61 (3), 539–542.
https://doi.org/10.1093/sysbio/sys029
Singer, R.A. & Page, L.M. (2015) Revision of the zipper loaches, Acanthocobitis and Paracanthocobitis (Teleostei: Nemachei-
lidae), with descriptions of five new species. Copeia, 103 (2), 378–401.
https://doi.org/10.1643/CI-13-128
Singer, R.A., Pfeiffer, J.M. & Page, L.M. (2017) A revision of the Paracanthocobitis zonalternans (Cypriniformes: Nemachei-
lidae) species complex with descriptions of three species. Zootaxa, 4324 (1), 85–107.
https://doi.org/10.11646/zootaxa.4324.1.5
Šlechtová, V., Bohlen, J. & Tan, H.H. (2007) Families of Cobitoidea (Teleostei; Cypriniformes) as revealed from nuclear genetic
data and the position of the mysterious genera Barbucca, Psilorhynchus, Serpenticobitis and Vaillantella. Molecular Phylo-
genetics and Evolution, 44, 1358–1365.
https://doi.org/10.1016/j.ympev.2007.02.019
Szllosi, G.J., Tannier, E., Daubin, V. & Boussau, B. (2013). The inference of gene trees with species trees. Systematic biology,
64 (1), 42–62.
https://doi.org/10.1093/sysbio/syu048
Zhang, D., Gao, F.L., Jakovlić, I., Zou, H., Zhang, J., Li, W.X. & Wang, G.T. (2020) PhyloSuite: an integrated and scalable
desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular
Ecology Resources, 20 (1), 348–355.
https://doi.org/10.1111/1755-0998.13096
Zhao, L.X., Liu, J.H., Du, L.N. & Luo, F.G. (2021) A new loach species of Troglonectes (Teleostei: Nemacheilidae) from
Guangxi, China. Zoological Research, 42 (4), 423–427.
https://doi.org/10.24272/j.issn.2095-8137.2021.028
Zhou, Y., Duvaux, L., Ren, G., Zhang, L., Savolainen, O. & Liu, J. (2017) Importance of incomplete lineage sorting and intro-
gression in the origin of shared genetic variation between two closely related pines with overlapping distributions. Heredity,
118 (3), 211–220.
https://doi.org/10.1038/hdy.2016.72
