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Resolving phylogenetic relationships and taxonomic revision in the Pseudogastromyzon (Cypriniformes, Gastromyzonidae) genus: molecular and morphological evidence for a new genus, Labigastromyzon

September 2023
Integrative Zoology 19(5):1-22

DOI:10.1111/1749-4877.12761

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CC BY-NC 4.0

Authors:

Jingchen Chen

Shanghai Ocean University

Wen-Qiao Tang

Shanghai Ocean University

Haotian Lei

China Agricultural University

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The Pseudogastromyzon genus, consisting of species predominantly distributed throughout southeastern China, has garnered increasing market attention in recent years due to its ornamental appeal. However, the overlapping diagnostic attributes render the commonly accepted criteria for interspecific identification unreliable, leaving the phylo-genetic relationships among Pseudogastromyzon species unexplored. In the present study, we undertake molecular phylogenetic and morphological examinations of the Pseudogastromyzon genus. Our phylogenetic analysis of mi-tochondrial genes distinctly segregated Pseudogastromyzon species into two clades: the Pseudogastromyzon clade and the Labigastromyzon clade. A subsequent morphological assessment revealed that the primary dermal ridge (specifically, the second ridge) within the labial adhesive apparatus serves as an effective and precise interspe-cific diagnostic characteristic. Moreover, the distributional ranges of Pseudogastromyzon and Labigastromyzon are markedly distinct, exhibiting only a narrow area of overlap. Considering the morphological heterogeneity of the labial adhesive apparatus and the substantial division within the molecular phylogeny, we advocate for the elevation of the Labigastromyzon subgenus to the status of a separate genus. Consequently, we have ascertained the validity of the Pseudogastromyzon and Labigastromyzon species, yielding a total of six valid species. To facilitate future research, we present comprehensive descriptions of the redefined species and introduce novel identification keys.

Phylogenetic tree of the Pseudogastromyzon genus based on mitochondrial PCGs and rRNA genes, using Sinogastromyzon szechuanensis as an outgroup. Tree topology inferred from ML and BI methods with node support values (ML/BI).

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Phylogenetic tree of Pseudogastromyzon cheni, P. peristictus, P. laticeps, and P. lianjiangensis collected from coastal hills in southern Fujian to eastern Guangdong using haplotypes of mitochondrial cytb genes, with P. myersi as outgroups. The topology of phylogenetic tree was inferred from ML and BI methods. Bootstrap supports for each analysis are indicated at the nodes (ML/BI).

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a-c) Dorsal, lateral, and ventral views of Pseudogastromyzon fasciatus (topotypic), P. fasciatus jiulongjiangensis (topotypic), and P. meihuashanensis (Datian country). (d-f) Labial adhesive apparatus for each species. (g) Schematic of unimodal waveshaped apparatus, marking ridge positions; second ridge is primary with a single obtuse hump beyond fourth ridge, others are secondary; central ridge highly variable.

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a,b) Dorsal, lateral, and ventral views of Pseudogastromyzon cheni (topotypic) and P. peristictus (topotypic), respectively. (c,d) Labial adhesive apparatus for each species. (e) Schematic of unimodal wave-shaped apparatus, marking ridge positions; second ridge is primary with a single acuminate hump beyond fourth ridge, others are secondary; central ridge highly variable.

…

a,b) Dorsal, lateral, and ventral views of Pseudogastromyzon laticeps (topotypic) and P. lianjiangensis (topotypic). (c,d) Labial adhesive apparatus of P. laticeps and P. lianjiangensis, respectively. (e,f) The relative positions of the pectoral fin origin and the eye for P. laticeps and P. lianjiangensis are depicted, both exhibiting a high degree of variability. (g) A condition in P. lianjiangensis is illustrated, where the scaleless abdominal region extends up to the vicinity of the pelvic fin base. (h) Schematic diagram of bimodal wave-shaped labial adhesive apparatus, marking ridge positions; second ridge is primary with a dual hump beyond fourth ridge, others are secondary; central ridge highly variable.

…

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Integrative Zoology 2023; 0: 1–22 doi: 10.1111/1749-4877.12761

ORIGINAL ARTICLE

Resolving phylogenetic relationships and taxonomic revision in

the Pseudogastromyzon (Cypriniformes, Gastromyzonidae) genus:

molecular and morphological evidence for a new genus,

Labigastromyzon

Jingchen CHEN,1,2 Yiyu CHEN,3Wenqiao TANG,1,2 Haotian LEI,4Jinquan YANG1, 2

and Xiaojing SONG1,2

1Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, Shanghai, China,

2Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University,

Shanghai, China, 3National Natural Science Foundation of China, Beijing, China and 4College of Resources and Environment

Sciences, China Agricultural University, Beijing, China

Abstract

The Pseudogastromyzon genus, consisting of species predominantly distributed throughout southeastern China, has

garnered increasing market attention in recent years due to its ornamental appeal. However, the overlapping diag-

nostic attributes render the commonly accepted criteria for interspeciﬁc identiﬁcation unreliable, leaving the phylo-

genetic relationships among Pseudogastromyzon species unexplored. In the present study, we undertake molecular

phylogenetic and morphological examinations of the Pseudogastromyzon genus. Our phylogenetic analysis of mi-

tochondrial genes distinctly segregated Pseudogastromyzon species into two clades: the Pseudogastromyzon clade

and the Labigastromyzon clade. A subsequent morphological assessment revealed that the primary dermal ridge

(speciﬁcally, the second ridge) within the labial adhesive apparatus serves as an effective and precise interspe-

ciﬁc diagnostic characteristic. Moreover, the distributional ranges of Pseudogastromyzon and Labigastromyzon are

markedly distinct, exhibiting only a narrow area of overlap. Considering the morphological heterogeneity of the

labial adhesive apparatus and the substantial division within the molecular phylogeny, we advocate for the elevation

of the Labigastromyzon subgenus to the status of a separate genus. Consequently, we have ascertained the validity

of the Pseudogastromyzon and Labigastromyzon species, yielding a total of six valid species. To facilitate future

research, we present comprehensive descriptions of the redeﬁned species and introduce novel identiﬁcation keys.

Key words: Labigastromyzon, mitochondrial genome, phylogeny, Pseudogastromyzon, taxonomy

Correspondence: Wenqiao Tang, Shanghai Universities Key

Laboratory of Marine Animal Taxonomy and Evolution,

Shanghai Ocean University, Shanghai 201306, China.

Email: wqtang@shou.edu.cn

INTRODUCTION

Pseudogastromyzon, a unique group of ﬁshes inhab-

iting swift-ﬂowing mountain streams, possess special-

ized body structures, like complex labial adhesive appa-

ratus, that facilitate adhesion to rocks. In recent years, the

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J. Ch en et al.

captivating appearance of these ﬁsh has led to an uptick

in their capture from the wild, resulting in their increased

presence in commercial markets. Taxonomically, Pseudo-

gastromyzon falls under Actinopteri, Cypriniformes, and

Gastromyzontidae.

The inaugural formal publication of the Pseudogas-

tromyzon species occurred in 1878, based on specimens

procured from Wuyishan County, Fujian. Sauvage (1878)

initially classiﬁed it as a novel species within the genus

Psilorhynchus, dubbing it Psilorhynchus fasciatus, albeit

without specifying the rationale for this particular classi-

ﬁcation.

In 1925, Nichols discovered a new ﬁsh species in

the neighboring Nanping County, considering it a novel

Hemimyzon species and naming it Hemimyzon zebroidus.

He observed that this species exhibited a markedly re-

duced number of pelvic ﬁn rays compared to Hemimyzon

formosana (Boulenger 1894) and possessed an obliquely

truncate caudal ﬁn, in contrast to the forked caudal ﬁn

of the latter. Based on these attributes, Nichols catego-

rized Hemimyzon zebroidus as a new subgenus, Pseudo-

gastromyzon (Nichols 1925).

In 1930, Fang promoted Pseudogastromyzon to the

genus rank, highlighting the presence of a single barbel

on each angle of the mouth, the absence of an indenta-

tion between the lower jaw and snout, and a skin ﬂap at

the base of the pelvic ﬁns as distinguishing features that

set it apart from Hemimyzon. Moreover, Fang posited that

Pseudogastromyzon represented a less specialized group

compared to Beaufortia (Fang 1930).

Upon comparing the specimens of Psilorhynchus fas-

ciatus and Hemimyzon zebroidus, Hora (1931, 1932)

reclassiﬁed Psilorhynchus fasciatus under the genus

Pseudogastromyzon and considered Pseudogastromyzon

zebroidus a synonym. Hora also regarded Pseudogas-

tromyzon as the ancestral form of Beaufortia, sharing a

close genetic relationship.

In Fang’s (1933) research, he further elucidated the

differences between Pseudogastromyzon and the genera

Beaufortia and Sewellia, and emphasized the greater spe-

cialization of Pseudogastromyzon’s mouth compared to

Beaufortia. In the same study, Fang reclassiﬁed Crossos-

toma fangi (Nichols 1931) under the Pseudogastromyzon

genus.

In 2001, Kottelat assigned four species documented in

Vietnam—Gastromyzon buas (Mai 1978), Gastromyzon

daon (Mai 1978), Gastromyzon elongata (Mai 1978), and

Gastromyzon loos (Mai 1978)—to the genus Pseudogas-

tromyzon based on the presence of a notched pelvic disc

(Kottelat 2001). However, it is crucial to note that the

ventral ﬁns of Pseudogastromyzon species are entirely

separate and do not form a complete pelvic disc, and the

lower lip has evolved into a distinct labial adhesive ap-

paratus, setting them apart from other genera. Kottelat

(2012, 2013) subsequently reevaluated the classiﬁcation

of these four species, reassigning them to the genus Beau-

fortia. Consequently, these speciﬁc species are not perti-

nent to the current study.

As it stands, Fishbase statistics reveal that there are

nine valid Pseudogastromyzon species, excluding the

aforementioned four (Froese & Pauly 2010). The identi-

ﬁcation of these species primarily hinges on the overall

morphology and proportional ranges of speciﬁc body

parts. However, this approach is vulnerable to various

factors, such as growth stages, individual developmental

differences, sexual dimorphism, regional population

variations, specimen preservation status, and measure-

ment errors. The growing collection of specimens from

diverse regions has exacerbated the issues associated with

employing the aforementioned identiﬁcation methods.

Instances where molecular identiﬁcation is possible, but

morphological distinctions are insufﬁcient, have arisen.

This poses signiﬁcant challenges to subsequent research

and resource development efforts for species within this

genus.

In light of these concerns, the present study aims to

analyze the reliability of all major previously identiﬁed

characteristics for species identiﬁcation within the genus

and explore new, more effective morphological identiﬁ-

cation features. Furthermore, this research employs mito-

chondrial genomes to construct a phylogenetic tree within

the genus Pseudogastromyzon, examining the phyloge-

netic relationships among species. By integrating molec-

ular phylogenetics and morphological characteristics, we

seek to reevaluate and reorganize the classiﬁcation of

species within the genus.

MATERIALS AND METHODS

Field survey and sample collection

Sampling locations are shown in Fig. 1 and Table 1.

Nine species and two subspecies of Pseudogastromyzon

were collected for mitochondrial genome (mt-genome)

sequencing. P. fasciatus,P. cheni,P. laticeps,P. lian-

jiangensis,P. changtingensis,P. fangi, and P. fascia-

tus jiulongjiangensis were collected from their type lo-

calities; others were collected from nearby locations

due to habitat changes or inconsistencies with original

descriptions.

In addition, we used separate collections of P. laticeps,

P. lianjiangensis,P. cheni, and P. peristicus for mitochon-

drial cytochrome b gene sequencing and phylogenetic

analysis to determine the validity of the aforementioned

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Phylogeny of Pseudogastromyzon

Figure 1 Pseudogastromyzon species distribution map by county. Shading indicates species presence, with overlapping lines for

co-occurring species. Species denoted by letters in yellow circles are detailed in Table 1.

species. All specimens were euthanized immediately after

collection, preserved in 95% alcohol, and stored in a 4°C

refrigerator in the laboratory.

Distribution maps were created based on this study

and previous research (Pan et al. 1984; Song 1992; He

et al. 2000; Li & Xie 2004; Li et al. 2007; Huang et al.

2009; Li & Lin 2010; Liu et al. 2020; Yuan et al. 2020;

Xiao et al. 2021).

All ﬁsh collection followed the Law of the People’s

Republic of China on the Protection of Wildlife, and

we strictly adhered to all applicable international, na-

tional, and institutional guidelines for the care and use of

animals.

Specimen measurement and morphological

comparison

In previous studies (Zheng & Chen 1980; Chen 1980;

Zheng & Li 1986; Li 1998), morphological characteristics

used for species identiﬁcation included the morphology

of the labial adhesive apparatus, the ratio of head height

to head width at the origin of the pectoral ﬁn (PHW/

PHH), whether the origin of the pectoral ﬁn extends

beyond the vertical line through the middle of the eye,

the ratio of caudal peduncle length to caudal peduncle

height (CPL/CPH), the number of lateral stripes, and

the size of lateral circular spots. This study observed,

measured, and compared these features to discuss their

signiﬁcance in differentiating species. Specimens were

initially identiﬁed using original literature, and molec-

ular determination was conducted for unidentiﬁable

specimens. The species that require discrimination using

PHW/PHH and CPL/CPH were P. laticeps,P. lianjiangen-

sis,P. cheni,P. peristicus, and P. myersi. Measurements

were taken with a dial caliper and recorded to the nearest

0.01 mm. Normality tests and one-way ANOVA were per-

formed on the data using SPSS v.26.0. The labial adhesive

apparatus was observed using a stereoscopic microscope.

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J. Ch en et al.

Tab l e 1 Sample collection and sequencing information of this study.

Label Species Sampling location GenBank accession Sequence type

aPseudogastromyzon fasciatus

fasciatus

Wuyishan County, Fujian Province MH285818 mt-genome

bP. meihuashanensis Datian County, Fujian Province MH285822 mt-genome

cP. fasciatus jiulongjiangensis Nanjing County, Fujian Province MH285819 mt-genome

d1 P. c h e n i Changting County, Fujian Province MH285816 mt-genome

d2 P. c h e n i Shanghang County, Fujian Province OP950824 mt-cytb

d3 P. c h e n i Longyan County, Fujian Province OP950825 mt-cytb

e1 P. peristictus Wuping County, Fujian Province MH285824 mt-genome

e2 P. peristictus Jiaoling County, Guangdong

Province

OP950826 mt-cytb

f1 P. laticeps Haifeng County, Guangdong

Province

MH285820 mt-genome

f2 P. laticeps Haifeng County, Guangdong

Province

OP950827 mt-cytb

f3 P. laticeps Shanwei City, Guangdong Province OP950828 mt-cytb

f4 P. laticeps Luhe County, Guangdong Province OP950829 mt-cytb

f5 P. laticeps Jiexi Counth, Guangdong Province OP950830 mt-cytb

f6 P. laticeps Puning County, Guangdong Province OP950831 mt-cytb

g1 P. lianjiangensis Puning County, Guangdong Province MH285821 mt-genome

g2 P. lianjiangensis Puning County, Guangdong Province OP950832 mt-cytb

hP. m y e r s i Shenzhen City, Guangdong Province MH285823 mt-genome

iP. changtingensis changtingensis Changting County, Fujian Province MH271100 mt-genome

jP. changtingensis tungpeiensis Shicheng County, Jiangxi Province MH271101 mt-genome

kP. fangi Yingde County, Guangdong Province MH285817 mt-genome

Mitochondrial genes sequencing

The DNA was extracted from the right pectoral ﬁn tip

with the kit produced by Sangon Biotech (Shanghai) Co.,

Ltd. according to the steps in the manual. The cytb gene

was ampliﬁed using primers GAC TTG AAG AAC CAC

CGT TGT TAT T 5’−3’ and TCT TCG GAT TAC AAG

ACCGATGCTTT5’−3’. The PCR system contains 1

µL DNA template, 1 µL each primer (10 mol L−1), 12.5

µL Taq Mix (Sangon Biotech), and 9.5 µLddH

2O, for a

total reaction volume of 25 µL. The PCR reaction condi-

tions included a pre-denaturation step at 95°C for 3 min,

denaturation at 94°C for 30 s, annealing at 55°C for 45 s,

extension at 72°C for 60 s, and 35 cycles of ampliﬁcation.

This was followed by a ﬁnal extension step at 72°C for

5 min and a holding step at 4°C. The ampliﬁed products

were then puriﬁed and sequenced by Sangon Biotech.

For mitochondrial genome sequencing, DNA was

extracted using the CTAB method and its quality was

assessed. Subsequently, the DNA was fragmented using

ultrasonic waves, followed by fragment puriﬁcation,

terminal repair, 3’ end addition of A, and joining of

sequencing adapters. Fragment size selection was car-

ried out using agarose gel electrophoresis, and the

resulting fragments were ampliﬁed via PCR to create

a sequencing library, using the NEBNext® UltraTM

DNA Library Prep Kit for Illumina®. The prepared

library was subjected to agarose gel electrophoresis

for fragment selection, and only the qualiﬁed library

proceeded to sequencing using the Illumina NovaSeq

platform at a depth of 100X. The original image data

ﬁle was then converted to raw data via base calling

analysis. Reads with N base content greater than 5%,

reads with more than 50% low quality bases (quality

value ≤5), and reads with adapter contamination were

removed using SOAPnuke v. 1.3.0 (Chen et al. 2018).

The genome was spliced using SPAdes v. 3.13.0 (Banke-

vich et al. 2012), and the resulting spliced sequences

were compared to related reference genomes (GenBank

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Phylogeny of Pseudogastromyzon

accession: AP013300) using blastn (version: blast

2.2.30+; Chen et al. 2018 parameters: -evaluate 1 ×

10−5). Based on the alignment, the candidate sequence

assembly results were determined. In case the assembled

sequence contains gaps (including N sequences), the gaps

were ﬁlled using Gapcloser (Xu et al. 2020) to obtain the

ﬁnal splicing result. The gene structure was annotated

using MITOS (Donath et al. 2019), an online annota-

tion software designed speciﬁcally for mitochondrial

genomes. The genome annotations were entered on the

MITOS homepage.

Genetic distances estimates and phylogenetic

analyses

We utilized MEGA-11 (Tamura et al. 2021) and

the Kimura-2-parameter method to estimate genetic dis-

tances between mitochondrial genomes and nucleotide se-

quences of protein-coding genes (PCGs).

Outgroups included two Erromyzon (KY352769,

MH155188), two Liniparhomaloptera (AP013301,

MZ047229), and one Sinogastromyzon (MN241814,

Balitoridae) from the NCBI database. The 12S, 16S

rRNA gene, and 13 PCGs of all samples were extracted,

aligned (using Clustal W), and concatenated in their

genomic arrangement order utilizing PhyloSuite (Zhang

et al. 2020).

Substitution saturation was tested using DAMBE (Xia

& Xie 2001), yielding Iss: 0.1397 <Iss.c: 0.8530 and

Prob (two-tailed) =0, indicating dataset suitability for

phylogenetic analysis. PartitionFinder2 (Lanfear et al.

2017) determined the optimal substitution model as GTR

+G+I. A maximum likelihood phylogenetic tree was

constructed using IQtree (Nguyen et al. 2015) within Phy-

loSuite, with the GTR +G+I model, ultrafast bootstrap

(5000 replicates, Hoang et al. 2018), and MN241814 as

the designated outgroup. A Bayesian phylogenetic tree

was constructed using the same partition model and the

built-in Mrbayes (Ronquist & Huelsenbeck 2003) pro-

gram in PhyloSuite. Parameters included 1000 000 gen-

erations, print frequency of 1000, sample frequency of

100, four chains and runs, and check-pointing frequency

of 5000. Convergence reached below 0.01 at the run’s end.

The second dataset comprised mt-cytb gene sequences

from Genbank (MZ853164, MZ853158) and extracted

from various geotypes of P. laticeps,P. lianjiangensis,

P. cheni, and P. persiticus. MEGA-11 software was em-

ployed for sequence alignment. Built-in ModelFinder

(Kalyaanamoorthy et al. 2017) in IQtree determined the

best-ﬁt model as MG +F3 ×4+G4 for the ML method

and HKY +F+G4 for the BI method. The remaining

BI parameter settings were identical to those described

previously.

The ﬁnal tree was visualized and reﬁned using Figtree

v1.4.4 (Rambaut 2018).

Mitochondrial PCG divergence time estimation

and ancestral range reconstruction

The analysis incorporated mitochondrial PCG se-

quences from P. fasciatus,P. cheni,P. laticeps,P. m y -

ersi,P. changtingensis,P. fangi, and Erromyzon sinen-

sis (GenBank accession: MH155188). Divergence time

estimation was performed using BEAST 1.10 (Suchard

et al. 2018). Due to the lack of fossil records and un-

resolved evolutionary relationships, we concatenated 13

mitochondrial PCGs and applied a mutation rate of 0.02/

Ma (Xiao & Zhang 2000). We employed the GTR +G+

I model (Kalyaanamoorthy et al. 2017), an uncorrelated

lognormal relaxed clock, and the Rule Process. Bayesian

MCMC analyses ran for 400 million steps with results

veriﬁed in Tracer v1.7.2 (Rambaut et al. 2018). TreeAn-

notator v1.10.4 (Helfrich et al. 2018) was used to obtain

the tree ﬁle and Figtree for visualization.

Species distribution data delineated Pseudogastromy-

zon distribution into three regions: west of the Wuyi

Mountains and Jiulian Range (Region A); east of these

mountains, divided by the Ting River into southern (Re-

gion B) and northern (Region C) areas. Ancestral range

reconstruction was conducted using RASP (Yu et al.

2015) and BioGeoBEARS (Matzke 2013) for model test-

ing. The BAYAREALIKE model, with a maximum range

size of 2, was selected based on the highest AICc_wt

value (0.32). The output displayed only the most likely

ancestral states for each node in the phylogenetic tree.

RESULTS AND DISCUSSION

The distribution of Pseudogastromyzon

The distribution map is shown in Fig. 1. The west-

ernmost distribution of the Pseudogastromyzon species

reaches the central part of Guangxi Province, in Laibin

City, with the northern boundary lying south of the

Yangtze River. There is a clear division in the distribution

of each species. Among them, P. fangi and P. changtingen-

sis are widely distributed throughout the Nanling Moun-

tain Range. The Luoxiao Mountain Range is connected to

the Nanling Mountain Range and extends northward, so

these two species also have a widespread distribution in

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J. Ch en et al.

Tab l e 2 Length and base compositon of mitochondrial DNA sequence of Pseudogastromyzon.

Base composition (%)

Species Length (bp) A T G C A +TG+C

P. fasciatus fasciatus 16 561 29.71 25.20 16.51 28.57 54.91 45.09

P. fasciatus jiulongjiangensis 16 560 29.72 25.27 16.47 28.54 54.98 45.02

P. meihuashanensis 16 561 29.84 25.17 16.38 28.62 55.00 45.00

P. c h e n i 16 567 29.78 25.29 16.39 28.54 55.07 44.93

P. peristictus 16 566 29.83 25.21 16.33 28.64 55.03 44.97

P. laticeps 16 561 29.39 24.76 16.85 29.01 54.15 45.85

P. lianjiangensis 16 568 29.10 24.79 17.09 29.02 53.89 46.11

P. myersi 16 561 29.39 24.94 16.87 28.80 54.33 45.67

P. changtingensis changtingensis 16 571 29.27 25.48 16.96 28.28 54.76 45.24

P. changtingensis tungpeiensis 16 574 29.01 25.09 17.26 28.65 54.10 45.90

P. fangi 16 572 29.17 24.99 17.12 28.72 54.16 45.84

Mean 16 566 29.47 25.11 16.75 28.67 54.58 45.42

this area. However, they do not extend eastward beyond

the Wuyi Mountain Range. The overlapping distribution

area between these two species and the remaining species

is limited to the Dongjiang River Basin and the surround-

ing mountainous regions. P. fasciatus is widely distributed

in the Wuyi Mountain Range and its eastern side, extend-

ing to the coastal mountains. The remaining species are

mainly concentrated in the coastal mountainous areas of

eastern Guangdong Province.

Mitochondrial genome characteristics in

Pseudogastromyzon species

The mitochondrial genome sequences of Pseudogas-

tromyzon species range from 16 560 to 16 574 base

pairs. The base composition consists of A (29.47%), T

(25.11%), G (16.75%), and C (28.67%). The A +T con-

tent (54.58%) is notably higher than the G +C con-

tent (45.42%). Each sequence exhibits an A +T con-

tent greater than 50%, while the G content is signiﬁcantly

lower than that of C, indicating a distinct A +T prefer-

ence and anti-G bias (refer to Table 2).

All Pseudogastromyzon species possess circular mito-

chondrial genomes with a consistent gene arrangement,

including 13 PCGs, 2 rRNA genes, 22 tRNA genes, and

1 major non-coding control region (D-loop). With the ex-

ception of the ND6 gene and eight tRNA genes (tRNA-

Gln, Ala, Asn, Cys, Tyr, Ser (TGA), Glu, and Pro) situated

on the light strand (L-strand), all other genes are located

on the heavy strand (H-strand).

Table 3 demonstrates that, aside from COI, which em-

ploys GTG as the initiation codon encoding valine (Val),

the other 12 genes utilize ATG as the starting codon, en-

coding methionine (Met). There are four types of termi-

nation codons: two complete stop codons (TAA, TAG)

and two incomplete stop codons (TA-, T-). Their us-

age frequency, in descending order, is TAA (35.66%), T-

(30.07%), TA- (23.08%), and TAG (11.19%).

A noteworthy feature is the six-base-pair insertion after

base 1539 (corresponding to amino acid 513) in the COI

gene of P. changtingensis changtingensis,P. changtingen-

sis tungpeiensis, and P. fangi. The corresponding amino

acids for these two codons are Gln and either Ala (in P.

fangi and P. changtingensis tungpeiensis)orThr(inP.

changtingensis changtingensis).

Estimation of genetic distances

Upon evaluating Kimura’s two-parameter genetic

distances of mitochondrial PCGs and entire sequences,

we observed that the interspeciﬁc genetic distance among

P. fasciatus,P. fasciatus jiulongjiangensis, and P. m e i -

huashanensis is narrower compared to other species

(P. fasciatus vs. P. fasciatus jiulongjiangensis =2.89/

2.36, P. fasciatus vs. P. meihuashanensis =2.51/2.02,

P. fasciatus jiulongjiangensis vs. P. meihuashanensis =

2.64/2.19 [for PCGs/complete sequences]). Analogous

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Phylogeny of Pseudogastromyzon

Tab l e 3 The mean length, start codon, and stop codon of the protein-coding genes of genus Pseudogastromyzon.

Gene name Usage

frequency

(%)

ATP6 ATP8 COI COII COIII ND1 ND2 ND3 ND4 DN4L ND5 ND6 Cyt b

Mean length

(bp)

683 168 1553 691 785 975 1045 349 1382 297 1839 522 1140

Start codon

ATG 11 11 – 11 11 11 11 11 11 11 11 11 11 92.31

GTG – – 11 – – – – – – – – – – 7.69

Stop codon

TAA – 11 9 – – 7 – – – 11 10 3 – 35.66

TAG – – 2 – – 4 – 1 – – 1 8 – 11.19

TA- 11 – – – 11 – – – 11 – – – – 23.08

T– – – – 11 – – 11 10 – – – – 11 30.07

Tab l e 4 Interspecies genetic distance of Pseudogastromyzon based on mitochondrial PCGs (below diagonal) and complete

mitochondrial sequence (above diagonal) (%) in Kimura 2-parameter genetic distance analysis.

1234567891011

1P. fasciatus 2.36 2.02 5.01 5.05 4.69 5.15 6.61 13.61 13.55 12.94

2P. fasciatus jiulongjiangensis 2.89 2.19 5.00 4.97 4.65 5.16 6.63 13.73 13.68 12.90

3P. meihuashanensis 2.51 2.64 4.85 4.87 4.50 5.01 6.64 13.45 13.37 12.75

4P. c h e n i 6.34 6.17 6.01 2.73 5.29 5.75 6.92 13.50 13.50 12.90

5P. peristictus 6.41 6.17 6.02 3.47 5.21 5.73 7.09 13.59 13.63 12.95

6P. laticeps 5.67 5.51 5.32 6.64 6.52 4.94 6.58 13.39 13.38 12.97

7P. lianjiangensis 6.49 6.26 6.18 7.37 7.31 6.13 6.99 13.71 13.87 13.18

8P. m y e r s i 8.40 8.22 8.31 8.87 9.09 8.25 8.91 13.56 13.40 13.01

9P. changtingensis 16.12 16.20 15.78 16.04 16.18 15.90 16.22 16.35 5.30 9.74

10 P. changtingensis tungpeiensis 15.90 15.89 15.57 15.98 16.19 15.82 16.41 15.98 6.23 9.45

11 P. fangi 15.43 15.32 15.14 15.48 15.53 15.49 15.76 15.75 11.68 11.40

ﬁndings were obtained for P. cheni versus P. peristictus

(3.47/2.73) and P. laticeps versus P. lianjiangensis (6.13/

4.94). P. myersi exhibits genetic distinctiveness from all

congeneric species. The intraspeciﬁc genetic distance of

P. changtingensis,P. changtingensis tungpeiensis, and P.

fangi is more proximate compared to the remaining eight

species (refer to Table 4).

Mitochondrial rRNA-PCG nucleotide sequence

phylogenetic analysis

Utilizing maximum likelihood and Bayesian meth-

ods, two phylogenetic trees were generated, exhibiting

identical topologies. The 11 taxa, comprising both species

and subspecies, formed a monophyletic assemblage,

which further bifurcated into two distinct clades charac-

terized by considerable genetic divergence. Clade I en-

compasses P. fangi,P. changtingensis, and P. changtingen-

sis tungpeiensis, while Clade II incorporates the residual

eight taxa, with morphologically analogous species ag-

gregating in proximity.

Clade II can be further partitioned into four distinct

lineages: (1) P. fasciatus,P. fasciatus jiulongjiangensis,

and P. meihuashanensis;(2)P. cheni and P. peristictus;

(3) P. laticeps and P. lianjiangensis; and (4) P. myersi,

which represents the most basal divergence within Clade

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J. Ch en et al.

Figure 2 Phylogenetic tree of the Pseudogastromyzon genus based on mitochondrial PCGs and rRNA genes, using Sinogastromyzon

szechuanensis as an outgroup. Tree topology inferred from ML and BI methods with node support values (ML/BI).

II. The genera Erromyzon and Pseudogastromyzon ex-

hibit a sister-group afﬁliation and concurrently constitute

a monophyletic assemblage in the current phylogenetic

reconstruction (refer to Fig. 2).

Mitochondrial cytb sequences phylogenetic

analysis

Utilizing maximum likelihood and Bayesian methods,

a pair of congruent phylogenetic trees was constructed,

presenting analogous topological structures. Notably, P.

laticeps and P. lianjiangensis, originating from disparate

geographical locales, coalesced into a uniﬁed lineage.

Concurrently, P. cheni and P. peristictus constituted a sep-

arate, well-deﬁned lineage. Intriguingly, the P. peristictus

taxon exhibited a non-monophyletic conﬁguration (refer

to Fig. 3).

Analysis of morphometric characteristics

The statistical information and signiﬁcant differences

of PHW/PHH and CPL/CPD are shown in Table 5. Both

data sets exhibit homoscedasticity.

Zheng and Chen (1980) initially described P. laticeps,

noting that the head height at the pectoral ﬁn origin is

half the head width at the pectoral ﬁn origin (PHW/PHH

=2). In contrast, this ratio is less than 2 for P. myersi,P.

cheni,P. lianjiangensis (Zheng 1981), and P. peristictus

(Zheng & Li 1986). This difference was considered a pri-

mary diagnostic criterion. The Bonferroni post hoc test

for multiple comparisons revealed that while signiﬁcant

differences exist between P. laticeps and both P. cheni and

P. peristictus in terms of the PHW/PHH ratio, no signiﬁ-

cant differences were observed between P. laticeps and P.

lianjiangensis or P. myersi. Consequently, this feature is

unsuitable for distinguishing P. laticeps from P. lianjian-

gensis and P. myersi.

Another diagnostic criterion proposed by Zheng and

Chen (1980) was the relative position of the pectoral

ﬁn origin and the eye in P. laticeps, which extends be-

yond the vertical midline of the eye, unlike other species.

However, as illustrated in Fig. 6e,f, this feature is vari-

able in both P. laticeps and P. lianjiangensis, making it

unreliable for accurate species identiﬁcation. Similarly,

general head shape descriptors, such as “blunt head” or

“rounded blunt head,” are insufﬁcient for precise diagno-

sis (Fig. 6e,f).

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Phylogeny of Pseudogastromyzon

Figure 3 Phylogenetic tree of Pseudogastromyzon cheni,P. peristictus,P. laticeps,andP. lianjiangensis collected from coastal hills

in southern Fujian to eastern Guangdong using haplotypes of mitochondrial cytb genes, with P. m y e r s i as outgroups. The topology of

phylogenetic tree was inferred from ML and BI methods. Bootstrap supports for each analysis are indicated at the nodes (ML/BI).

The scaleless abdominal region of P. laticeps, accord-

ing to the original description, only reaches the vicin-

ity of the pelvic ﬁn origin, while it extends beyond the

pelvic ﬁn axil in other species. This feature can effectively

distinguish P. laticeps from all other species except P.

lianjiangensis. However, mucus presence can affect this

characteristic in practical applications, and variability has

been observed in P. lianjiangensis (Fig. 6g). Zheng did

not use this feature for species identiﬁcation in the origi-

nal description of P. lianjiangensis.

Zheng and Chen (1980) proposed that in P. laticeps,

the caudal peduncle length is marginally shorter than the

caudal peduncle height (CPL/CPD <1), identifying this

as a crucial characteristic to differentiate it from P. m y -

ersi and P. cheni. While describing the novel species P.

lianjiangensis, Zheng (1981) asserted that the CPL/CPD

of P. lianjiangensis is equal to 1. In the description of

the new species P. peristictus, Zheng and Li (1986) main-

tained that its CPL/CPD >1 and employed this as a dis-

tinguishing feature between P. peristictus and the closely

related species P. cheni. Based on our analysis, the CPL/

CPD range for P. laticeps is 0.98–1.54 (1.24 ±0.15);

the Bonferroni post hoc test for multiple comparisons re-

veals no signiﬁcant difference from P. lianjiangensis but

signiﬁcant differences from the other three species. The

CPL/CPD range for P. lianjiangensis is 1.13–1.47 (1.33 ±

0.09), exhibiting signiﬁcant differences solely with P. m y -

ersi. The CPL/CPD range for P. peristictus is 1.20–1.68

(1.42 ±0.15), with no signiﬁcant difference from P. cheni.

Consequently, the caudal peduncle length-to-height ratio

is insufﬁcient for accurately distinguishing these species.

Zheng and Li (1986) once claimed that P. peristic-

tus exhibits smaller lateral round spots, with a diameter

smaller than the eye, while P. cheni exhibits larger spots

with a diameter greater than the eye. However, our ob-

servations found irregularity in the size and shape of the

lateral round spots in both species. Furthermore, the com-

parison method between these irregular shapes and eye di-

ameter was not explicitly explained by Zheng. Given the

high variability and difﬁculty in quantiﬁcation, we con-

sider the size of lateral spots an unreliable distinguishing

feature.

Chen (1980) initially suggested that P. fasciatus jiu-

longjiangensis has 10–15 lateral stripes, while P. fasciatus

has 16–21 lateral stripes. Subsequently, Li (1998) de-

scribed the new species P. meihuashanensis, which is

primarily characterized by having nine lateral stripes.

However, upon further observation, the number of lateral

stripes in these three species is not as clearly distinguish-

able as previously thought, displaying a high degree of

overlap (Table 6). Therefore, the number of lateral stripes

is unsuitable as a diagnostic feature for these species.

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J. Ch en et al.

Figure 4 (a–c) Dorsal, lateral, and ventral views of Pseudogastromyzon fasciatus (topotypic), P. fasciatus jiulongjiangensis (topo-

typic), and P. meihuashanensis (Datian country). (d–f) Labial adhesive apparatus for each species. (g) Schematic of unimodal wave-

shaped apparatus, marking ridge positions; second ridge is primary with a single obtuse hump beyond fourth ridge, others are sec-

ondary; central ridge highly variable.

Labial morphology analysis

Tang and Chen (1996) previously employed electron

microscopy to investigate the morphological characteris-

tics of the lower lip labial adhesive apparatus in Pseudo-

gastromyzon species, identifying two distinct morpholog-

ical types: club-shaped and wave-shaped. Based on this

primary evidence, they suggested the consolidation of the

three club-shaped species (P. fangi,P. changtingensis, and

P. changtingensis tungpeiensis) into a novel subgenus,

Labigastromyzon.

In the present study, we conducted an extensive exam-

ination of the lower lip labial adhesive apparatus struc-

tures across all Pseudogastromyzon species. Excluding

species belonging to the subgenus Labigastromyzon,the

lower lip labial adhesive apparatus in other Pseudogas-

tromyzon species typically comprises four dermal ridges:

one primary dermal ridge (the second dermal ridge) and

three secondary dermal ridges (the ﬁrst, third, and fourth

dermal ridge).

The ﬁrst dermal ridge, occupying the lowest position,

curves downward near the lower lip’s center and breaks

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Phylogeny of Pseudogastromyzon

Tab l e 5 Signiﬁcance analysis of the differences in PHW/PHH and CPL/CPH ratios for Pseudogastromyzon laticeps,P. lianjiangensis,P. myersi, P. peristictus,andP.

cheni.

Bonferroni post hoc analysis (signiﬁcance)

No. Species Sample size PHW/PHH

Min–max (mean ±SD)

1 2 3 4 5 CPL/CPH

Min–max (mean ±SD)

1P. laticeps 15 1.64–2.32 (2.00 ±0.20) 0.881 0.000 0.004 0.002 0.98–1.54 (1.25 ±0.15)

2P. lianjiangensis 15 1.97–2.39 (2.14 ±0.13) 0.222 0.000 0.529 0.304 1.13–1.47 (1.33 ±0.09)

3P. myersi 15 1.64–2.06 (1.85 ±0.15) 0.150 0.000 0.106 0.197 1.29–1.76 (1.55 ±0.14)

4P. peristictus 15 1.55–2.19 (1.80 ±0.20) 0.017 0.000 1.000 1.000 1.20–1.68 (1.42 ±0.15)

5P. c h e n i 15 1.42–1.93 (1.63 ±0.14) 0.000 0.000 0.007 0.068 1.27–1.69 (1.44 ±0.13)

In the post hoc analysis, values >0.05 indicate no signiﬁcant difference between the two species (lower-left corner: PHW/PHH; upper-right corner: CPL/CPH).

at the median. Both ends of this ridge extend toward the

mouth corners, where they connect with the upper lip’s

margin (refer to Figs 4d–f,5c,d,6c,d,7c).

The second dermal ridge arcs upward near the lower

lip’s center, forming a species-speciﬁc structure. In P.

cheni and P. peristictus, this ridge creates a single hump

at the adhesive apparatus’s upper margin, which we des-

ignate as “unimodal” (see Fig. 5c–e). In P. laticeps and

P. lianjiangensis, the second dermal ridge arches up-

ward near the lower lip’s center and presents a down-

ward concavity at its apex, forming a dual hump, which

we term “bimodal” (see Fig. 6c,d,g). In P. fasciatus,P.

fasciatus jiulongjiangensis, and P. meihuashanensis,the

second dermal ridge adopts an upward arch near the

lower lip’s center, generating a single hump that falls

into the “unimodal” category but with a rounded appear-

ance (see Fig. 4d–g). In these species, the second dermal

ridge’s highest point frequently surpasses the fourth der-

mal ridge; however, in P. myersi, a gap or a small prolifer-

ative papilla often separates the single peak of the second

dermal ridge from the fourth dermal ridge (see Fig. 7b,c).

Situated between the second and fourth dermal ridges,

the third dermal ridge slopes upward toward the lower

lip’s center, ceasing upon encountering the arching sec-

ond dermal ridge. This ridge may occasionally be absent,

fragmented, or exhibit proliferation. It represents the most

variable of the four dermal ridges.

The fourth dermal ridge typically ascends and fuses

with the upward-arching second dermal ridge at the lower

lip’s center. In P. myersi, the fourth dermal ridge is pre-

dominantly ﬂat, and its upper margin lacks a hump (see

Fig. 7b,c).

The termini of the second, third, and fourth dermal

ridges do not reach the mouth corners (refer to Figs 4d–

f,5c,d,6c,d,7c).

The lower lip labial adhesive apparatus of the subgenus

Labigastromyzon encompasses three dermal ridges. The

ﬁrst dermal ridge breaks in the middle, with both ends not

extending to the mouth corners and not connecting with

the upper lip. Papillae are commonly present on the ridge.

The second dermal ridge exhibits three abrupt turns,

forming a vertical “M” shape, with both ends reaching

the mouth corners and curving upward. The third dermal

ridge is generally ﬂat, with both ends bending downward

and fusing with the two vertical peaks of the second

dermal ridge (refer to Fig. 8d–g). Occasionally, a notch

appears in the middle. Between the second and third

dermal ridges, a short proliferative ridge may emerge,

adhering closely to the second dermal ridge’s lateral

side.

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. 11

J. Ch en et al.

Tab l e 6 Countable characteristics data for Pseudogastromyzon.

Species Dorsal ﬁn Anal ﬁn Pectoral ﬁn Pelvic ﬁn Lateral scales Lateral stripes

P. fasciatus iii-7-8 ii-5 i-16-19 i-8-9 67–80 10–19

P. fasciatus jiulongjiangensis iii-7-8 ii-5 i-17-18 i-8-9 68–75 10–15

P. meihuashanensis iii-7-8 ii-5 i-17-18 i-8-9 68–74 10–14

P. c h e n i iii-7-8 ii-5 i-15-17 i-8-9 68–76 –

P. peristictus iii-7-8 ii-5 i-15-17 i-8-9 70–76 –

P. laticeps iii-7-8 ii-5 i-16-18 i-9 51–70 –

P. lianjiangensis iii-7 ii-5 i-17-18 i-9 63–67 –

P. myersi iii-7-8 ii-5 i-16-17 i-8-9 66–74 –

P. changtingensis iii-7-8 ii-5 i-16-17 i-7-8 71–76 14–15

P. changtingensis tungpeiensis iii-7-8 ii-5 i-15-17 i-7-8 69–76 12–17

P. fangi iii-8-10 ii-5 i-17-19 i-7-10 73–80 14–19

Taking into account the lower lip labial adhesive ap-

paratus structure and additional morphological features,

Pseudogastromyzon species can be categorized into the

following groups: the Labigastromyzon group, character-

ized by a club-shaped lower lip labial adhesive apparatus

and comprising P. changtingensis,P. changtingensis tung-

peiensis, and P. fangi;themyersi group, featuring a gener-

ally ﬂat fourth dermal ridge and the second dermal ridge

not surpassing the fourth dermal ridge, including P. m y -

ersi;thelaticeps group, possessing a bimodal second der-

mal ridge, including P. laticeps and P. lianjiangensis;the

cheni group, displaying a unimodal second dermal ridge

and irregular spots on the body side, including P. cheni

and P. peristictus; and the fasciatus group, showcasing

a rounded unimodal second dermal ridge and prominent

vertical stripes on the body side. This classiﬁcation corre-

sponds with molecular phylogenetic ﬁndings.

Taxonomic status promotion of subgenus

Labigastromyzon

Based on the ﬁndings of this study, we propose the el-

evation of Labigastromyzon from a subgenus to a distinct

genus, substantiated by the following evidence:

Geographical distribution: Labigastromyzon is pri-

marily concentrated in the Nanling-Luoxiao Mountain

Range and Yunkai Mountains Range. In contrast, Pseu-

dogastromyzon predominantly inhabits the Guangdong

southeast coastal hills and Wuyi Mountains Range, leav-

ing only a narrow overlapping area between them.

Genetic distance: Our analysis reveals a signiﬁcant ge-

netic distance between the Labigastromyzon and Pseu-

dogastromyzon species, surpassing the distance observed

between species within the same genus.

Phylogenetic analysis: Mitochondrial gene phyloge-

netic analysis demonstrates that the Labigastromyzon

species form a distinct clade, separate from the Pseudo-

gastromyzon clade. The branch length from the nearest

common ancestor of Labigastromyzon to the nearest com-

mon ancestor of both Labigastromyzon and Pseudogas-

tromyzon is considerably longer than other branches.

Morphological differences: Labigastromyzon exhibits

a unique labial adhesive apparatus structure, character-

ized by a club-shaped morphology, in contrast to the

wave-shaped morphology observed in Pseudogastromy-

zon. Notably, the ﬁrst adhesive apparatus dermal ridge in

Labigastromyzon does not connect or fuse with the up-

per lip, unlike in Pseudogastromyzon. Additionally, the

primary labial adhesive apparatus dermal ridge (second

dermal ridge) presents a standard inverted “M” shape in

Labigastromyzon, whereas Pseudogastromyzon displays a

unimodal or bimodal ridge.

Taxonomic research conventions: Zheng and Chen

(1980), Zheng (1981), Zheng and Li (1986), Li (1998),

and Chen and Tang (2000) employed the labial adhesive

apparatus shape (club-shaped or otherwise) as the primary

distinguishing characteristic when describing species. As

a result, these two groups have been consistently an-

alyzed separately in taxonomic research. Thus, elevat-

ing Labigastromyzon to genus status not only aligns

with morphological, phylogenetic, and biogeographical

theories but also adheres to the established classiﬁcation

research practices for this group.

The description of the genus Labigastromyzon and the

key to Labigastromyzon species are provided as follows:

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Phylogeny of Pseudogastromyzon

Figure 5 (a,b) Dorsal, lateral, and ventral views of Pseudogastromyzon cheni (topotypic) and P. peristictus (topotypic), respectively.

(c,d) Labial adhesive apparatus for each species. (e) Schematic of unimodal wave-shaped apparatus, marking ridge positions; second

ridge is primary with a single acuminate hump beyond fourth ridge, others are secondary; central ridge highly variable.

Labigastromyzon Tang and Chen, 1996

A slender type of hillstream suck loach characterized

by a cylindrical head and trunk, a ﬂattened ventral sur-

face, and a body that becomes progressively laterally

compressed posterior to the pelvic ﬁns. The pectoral ﬁn

tips cover the origin of the pelvic ﬁns, and the left and

right pelvic ﬁns are distinctly separated. The lower lip

exhibits a specialized, club-shaped labial adhesive appa-

ratus.

Remarks. Labigastromyzon can be distinguished

from other genera within the Gastromyzontidae family in

China by a unique combination of morphological traits:

the presence of a lower lip with adhesive apparatus, which

is also found in Pseudogastromyzon but absent in Lin-

iparhomaloptera,Vanmanemia,Formosania,Paraproto-

myzon,Beaufortia,Plesiomyzon,Erromyzon, and Yaosha-

nia; the pectoral ﬁn tips covering the pelvic ﬁn origin

and the specialization of pectoral and pelvic ﬁns as

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. 13

J. Ch en et al.

Figure 6 (a,b) Dorsal, lateral, and ventral views of Pseudogastromyzon laticeps (topotypic) and P. lianjiangensis (topotypic). (c,d)

Labial adhesive apparatus of P. laticeps and P. lianjiangensis, respectively. (e,f) The relative positions of the pectoral ﬁn origin and the

eye for P. laticeps and P. lianjiangensis are depicted, both exhibiting a high degree of variability. (g) A condition in P. lianjiangensis is

illustrated, where the scaleless abdominal region extends up to the vicinity of the pelvic ﬁn base. (h) Schematic diagram of bimodal

wave-shaped labial adhesive apparatus, marking ridge positions; second ridge is primary with a dual hump beyond fourth ridge, others

are secondary; central ridge highly variable.

disc-shaped sucker ﬁns, features shared by Pseudogas-

tromyzon,Paraprotomyzon, and Beaufortia, in contrast

to the pectoral ﬁn tips not reaching the pelvic ﬁn origin

in Liniparhomaloptera,Vanmanemia,Formosania,Ple-

siomyzon,Erromyzon, and Yaoshania.

Labigastromyzon can be further differentiated from

Pseudogastromyzon by the distinct shape of the labial ad-

hesive apparatus (club-shaped vs. wave-shaped).

Distribution. The genus Labigastromyzon is dis-

tributed throughout the southeastern coastal provinces

of China, including the hillstreams of the Pearl River

drainage in Guangdong and Guangxi provinces, the

Poyang Lake drainage in Jiangxi and Hunan provinces,

and the Ting River drainage in Fujian province.

The key to Labigastromyzon

1(2) Sides of body with 12–17 thick vertical stripes,

stripe width greater than the space between stripes

...............................L. changtingensis

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Phylogeny of Pseudogastromyzon

Figure 7 (a) Dorsal, lateral, and ventral views of Pseudogastromyzon myersi (Shenzhen). (b) Schematic diagram of peculiar wave-

shaped labial adhesive apparatus, marking ridge positions; the second ridge serves as a primary ridge, creating a single hump where

the peak does not surpass the fourth ridge, and the remaining ridges are secondary; central ridge highly variable. (c) Labial adhesive

apparatus of P. m y e r s i .

2(1) Sides of body densely covered with small spots or

thin stripes, stripe spacing greater than stripe width

.......................................L. fangi

Taxonomic revision

L. changtingensis and L. changtingensis tungpeiensis

In 1942, Liang identiﬁed P. fasciatus changtingensis,

which was later elevated to P. changtingensis by Chen

(1980) due to differences in the lower lip morphology. In

1949, Chen and Liang described P. tungpeiensis. Subse-

quently, Chen (1980) found it to be similar to P. changtin-

gensis but retained it as a valid subspecies, P. changtin-

gensis tungpeiensis. Chen (1980) distinguished it based

on mouth and head features, while Zheng and Li (1986)

observed fewer lateral stripes.

The present study reveals that individuals displaying

the phenotypic traits associated with a subspecies in ear-

lier research were found in almost all sampling loca-

tions, not exclusively in the type locality. Recognizing a

widely distributed form as a subspecies is not warranted.

Therefore, this study advocates for the elimination of the

subspecies status of P. changtingensis tungpeiensis,to

maintain rigor and avoid confusion in future research.

P. fasciatus,P. fasciatus jiulongjiangensis, and P.

meihuashanensis

In 1980, Chen described P. fasciatus jiulongjiangen-

sis as a novel subspecies of P. fasciatus, highlighting its

feature as comprising thicker lateral stripes on the body,

numbering between 10 and 15 (contrasted with thinner

stripes, ranging from 16 to 21). Subsequently, in 1998,

Li described P. meihuashanensis based on four specimens

procured from Zhangping County, Longyan City, Fujian

Province, asserting that the presence of 9 lateral stripes

set this species apart from related taxa (compared with

10–15 in P. fasciatus jiulongjiangensis and 16–21 in P.

fasciatus).

Our investigations reveal that taxa conforming to these

three descriptions are extensively distributed across the

Wuyi Mountain Range and its southern tributaries. The

number of stripes in P. fasciatus displayed considerable

variation, spanning from 10 to 19, while the other two taxa

exhibited distinct regional stripe counts. Mitochondrial

gene genetic distance analysis and phylogenetic data

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. 15

J. Ch en et al.

Figure 8 (a–c) Dorsal, lateral, and ventral views of Pseudogastromyzon fangi (topotypic), P. changtingensis (topotypic), and P.

changtingensis tungpeiensis (Youxian County), with P. fangi additionally showcasing another common pattern phenotype at the bot-

tom (specimen from Lingui County). (d–f) Labial adhesive apparatus of each species. (g) Schematic representation of the club-shaped

labial adhesive apparatus, with ridge positions labeled; the second ridge serves as a primary ridge, creating an upright “M” shape.

reveal that the genetic disparities between P. fasciatus,

P. fasciatus jiulongjiangensis, and P. meihuashanensis

are markedly smaller than those observed among other

species. Moreover, the phylogenetic outcomes corrobo-

rate their monophyletic nature, with abbreviated branch

lengths implying minimal genetic divergence and recent

separation. Consequently, P. fasciatus jiulongjiangensis

and P. meihuashanensis embody individual variations or

regional phenotypes of P. fasciatus. As such, P. m e i -

huashanensis is considered synonymous with P. fasciatus.

Given that the subspeciﬁc attributes of P. fasciatus jiu-

longjiangensis prevail in most populations, the subspecies

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Phylogeny of Pseudogastromyzon

designation of P. fasciatus jiulongjiangensis is rescinded

to circumvent potential confusion in the future.

Incorporating the studies of Sauvage (1878), Nichols

(1925), Chen (1980), Li (1998), and Chen and Tang

(2000), we have updated the description of P. fasciatus

as follows:

Pseudogastromyzon fasciatus (Sauvage, 1878)

Psilorhynchus fasciatus Sauvage, 1878, Bulletin de la

Société philomathique de Paris, 2(7):88 (Wuyishan Coun-

try, Fujian Province).

Hemimyzon zebroidus Nichols, 1925 American Mu-

seum Novitates, (167):1 (Nanping Country, Fujian

Province).

Pseudogastromyzon fasciatus jiulongjiangensis Chen,

1980, Acta Hydrobiologica Sinica, 7(1):110 (Longyan

Country and Nanjing Country, Fujian Province).

Pseudogastromyzon meihuashanensis Li, 1998, Jour-

nal of Fisheries of China, 22(7):260 (Zhangping Country,

Fujian Province).

Dorsal iii-7-8, anal ii-5, pectoral i-16-19, pelvic i-8-9;

lateral scales 67–80.

Head–thorax cylindrical, ﬂattened ventrally; body

compressed from pelvic ﬁn to caudal peduncle. Head

shorter than caudal ﬁn; eyes, nostrils dorsally situated;

eye diameter ∼equals gill slit; nostril ﬂap present, smaller

than eye. Snout with star-like protuberance, smaller than

eye. Lips and mouth ventrally situated, arcuate. Four ros-

tal barbels present; rostral lobes interspaced, 3–6 pro-

jections upon each margin; barbels and lobes shorter

than eye; inner layer adhered to upper jaw, connected

to lower lip at corner of mouth. Lower lip with spe-

cialized, wave-shaped adhesive apparatus consisting of

four dermal ridges. First dermal ridge runs along lower

lip, connected to upper lip at both ends; second ridge

curved, forming blunt single peak at adhesive appara-

tus center; third ridge between second and fourth. Inde-

terminate short proliferative dermal ridges between the

ﬁrst and second ridges, paired and oblique. One pair

of maxillary barbels present, shorter than eye. Dorsal

gill slit, small, ∼equals eye diameter. Scales smaller

than eye; head and thorax ventrally scaleless; lateral

line complete. Pectoral ﬁn origin ∼opposed to poste-

rior margin of eye; paired ﬁn rays extended, forming

disc-like structure; pectoral ﬁn tip covering pelvic ﬁn ori-

gin. Pelvic ﬁn origin situated ∼anterior 1/3 total length;

dorsal ﬂap at base, covering pelvic ﬁn base dorsally;

pelvic ﬁns separated. Dorsal ﬁn origin ∼opposed to pelvic

ﬁn, length of base not exceeding caudal ﬁn; ﬂattened

ends ∼opposed pelvic ﬁn tip. Anal ﬁn origin situated

∼posterior 1/3 total length, ends near caudal ﬁn origin.

Caudal ﬁn obliquely truncate, rays ventrally longer; cau-

dal ﬁn length ∼equal to the distance between the anal ﬁn

origin and caudal ﬁn base. Anus situated ∼posterior 1/3

between the pelvic and anal ﬁn origins. Note: "˜" repre-

sents an approximate equivalent in length or position.

Coloration. In life, dorsal body gradient from pale

yellow to deep brown, transitioning to white ventrally.

Head with deep brown spots, interspaced deep brown sad-

dle markings along the dorsal midline, 9–21 deep brown

vertical stripes on the sides, width equal or larger than

eye diameter. Fins with 2–6 rows of small black dots; ﬁn

membrane is translucent or semitransparent white. Dor-

sal ﬁn margin occasionally exhibits nuptial colors such as

deep black or brick red.

In alcohol solution, body dorsum is light to dark brown,

ventral white or pale yellow. In formalin solution, body

dorsum is light to dark brown, ventral yellow or dark

yellow.

Distribution. The species is extensively distributed

across the Wuyi Mountain range and the coastal moun-

tain streams situated to the east of the range.

Remarks. P. fasciatus can be differentiated from its

congeneric species based on the following morphologi-

cal traits: The second dermal ridge on the adhesive appa-

ratus displays a unimodal conﬁguration (in contrast to a

bimodal conﬁguration in P. laticeps); the lateral aspects

of the body exhibit 10–19 distinct deep brown vertical

stripes (as opposed to irregularly sized and shaped round

spots in P. cheni; alternatively, irregular coarse horizontal

bands and blotches interspersed along the lateral line in P.

myersi).

P. cheni and P. peristictus

Zheng and Li (1986) delineated the novel species, P.

peristictus, utilizing specimens procured from Fengshun

County, Guangdong Province. Upon examining 15 spec-

imens, the author distinguished the primary dissimilari-

ties between this species and its closely related congener,

P. cheni, as follows: body lateral spots with a diameter

smaller than that of the eye, and a caudal peduncle height

exceeding its length, in contrast to body lateral spots equal

to or larger than the eye diameter and a caudal peduncle

length surpassing its height in P. cheni.

The present study revealed that the discrepancies in

caudal peduncle dimensions (length and height) between

the two species lack signiﬁcance. Furthermore, the mag-

nitude and density of the body lateral spots display

considerable variability, thereby rendering them ineffec-

tive as discriminative characteristics. The genetic diver-

gence between P. peristictus and P. cheni is substantially

smaller than that between either species and other taxa.

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. 17

J. Ch en et al.

Phylogenetic analyses illustrate both species coalescing

within a monophyletic clade, while the “small round spot”

assemblage is evidenced to be paraphyletic. The geo-

graphic distribution of these two species spans from the

southern region of Changting County to Jiexi County,

exhibiting extensive overlap in their respective ranges.

Based on the cumulative evidence, P. peristictus is pro-

posed to embody a regional phenotype or individual vari-

ation of P. cheni, thereby warranting its classiﬁcation as a

synonym of P. cheni.

Incorporating the studies of Chen and Liang (1942),

Chen (1980), Zheng and Li (1986), and Chen and Tang

(2000), we have updated the description of P. cheni as fol-

lows:

Pseudogastromyzon cheni (Chen & Liang, 1942)

Pseudogastromyzon peristictus Zheng and Li, 1986,

Journal of Science and Medicine of Jinan University,

(7):77 (Fengshun Country, Guangdong Province).

Dorsal iii-7-8, anal ii-5, pectoral i-15-17, pelvic i-8-9;

lateral scales 68–76.

Body closely resembles P. fasciatus in general shape

and structure, but peak of the second dermal ridge of the

adhesive apparatus acuminate.

Coloration. In life, dorsal body gradient from light

gray, pale yellow to black, transitioning to white ventrally.

Head dorsal with deep brown spots, deep brown saddle

markings along dorsal midline, and irregular deep brown

circular spots densely distributed on sides. Fins with 2–

6 rows of small black dots; ﬁn membrane is translucent

or semitransparent white. Dorsal ﬁn margin occasionally

exhibits nuptial colors such as deep black or brick red.

Color changes in alcohol and formalin solutions con-

sistent with those of P. fasciatus.

Distribution. Observed in mountain streams span-

ning from Changting County to Jieyang County.

Remarks. P. cheni can be distinguished from con-

generic species with the following characteristics: the

apex of the second dermal ridge surpasses the fourth, with

the fourth dermal ridge distinctively acclivous; (vs. not

surpassing the fourth, with the fourth dermal ridge gen-

erally being ﬂat in P. myersi). The second dermal ridge

is unimodal (vs. bimodal in P. laticeps). The sides of the

body display irregularly sized and shaped round spots (vs.

10–19 distinct deep brown vertical stripes in P. fascia-

tus; or irregular, coarse horizontal bands and blotches dis-

persed along the lateral line in P. myersi).

P. laticeps and P. lianjiangensis

Zheng (1981) described the new species P. lianjian-

gensis based on specimens collected from Puning County,

and noted that the distinguishing features of this species

from the closely related P. laticeps are: the head height is

greater than half of the head width at the pectoral ﬁn ori-

gin; the pectoral ﬁn origin is approximately vertically be-

low the posterior margin of the eye (vs. head height equal

to half of the head width at the pectoral ﬁn origin, pectoral

ﬁn origin surpassing the vertical line through the center of

the eye in P. laticeps).

Our investigations reveal that the relative positioning

of the pectoral ﬁn origin and the eye among these two

species exhibits considerable variability. Moreover, there

is no signiﬁcant difference in their PHW/PHH ratios.

The genetic distance between the two is smaller than the

genetic distance between each and other species. Phy-

logenetically, the two species constitute a monophyletic

clade.

The distribution of P. laticeps spans coastal streams

from Shenzhen to Puning County and Jieyang County.

Within this range, the species manifests subtle morpho-

logical variations that do not strictly conform to its orig-

inal description. In a restricted sense, P. lianjiangensis is

conﬁned to Puning County, which demarcates the north-

eastern boundary of the P. laticeps distribution; there-

fore, the specimens from this region can be classiﬁed as

a regional morphotype of P. laticeps. The morphological

distinctions initially observed were predicated on nega-

tive premises, including insufﬁcient sampling, inadequate

specimen comparisons, and subjective observations in-

ﬂuenced by individual biases. Examples of these biases

include variations in head shape (round vs. square) and

the relative positioning of the pectoral ﬁn origin concern-

ing the eye’s center and posterior margin, which are sus-

ceptible to observer subjectivity.

Consequently, following extensive sampling and com-

prehensive analyses, we assert that P. lianjiangensis

should be regarded as a junior synonym of P. laticeps.

Incorporating the studies of Zheng and Chen (1980),

Chen (1980), Zheng (1981), and Chen and Tang (2000),

we have updated the description of P. laticeps as

follows:

Pseudogastromyzon laticeps (Chen & Zheng, 1980)

Pseudogastromyzon lianjiangensis Zheng, 1981, Jour-

nal of Jinan University (Natural Science), 1:59 (Puning

Country, Guangdong Province).

Dorsal iii-7-8, anal ii-5, pectoral i-16-18, pelvic i-9;

lateral scales 51–70.

Body closely resembles P. fasciatus in general shape

and structure, but the second dermal ridge arches up near

the center of the lower lip and indents at the median, form-

ing a dual hump at the upper margin of the adhesive ap-

paratus.

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Phylogeny of Pseudogastromyzon

Coloration. In life, dorsal body gradient from pale

yellow to black, transitioning to white ventrally. Head

dorsal with deep brown spots, or spots fused into worm-

like markings. Interspaced saddle markings along the dor-

sal midline. Signiﬁcant regional variation in populations,

primarily in lateral patterns and ﬁn colors. Lateral sides

are pattern-less, with small spots, and irregular markings

along the lateral line, or ﬁne vertical crossbars. Fins with-

out spots or with 2–6 rows of deep brown dots. Fin mem-

brane is translucent or exhibits nuptial colors such as pur-

ple, red, blue, and yellow. Fin margin presenting nuptial

colors such as black and blue.

In alcohol and formalin solutions, all nuptial colors

gradually disappear; remaining color changes similar to

those of P. fasciatus.

Remarks. P. laticeps can be differentiated from con-

generic species with the following characteristics: top

of the second dermal ridge exceeding the fourth, fourth

dermal ridge distinctively acclivous; (vs. top of the sec-

ond dermal ridge not exceeding the fourth, fourth dermal

ridge generally ﬂat in P. myersi). Second dermal ridge is

bimodal (vs. second dermal ridge unimodal in P. cheni

and P. fasciatus).

The key to renewed Pseudogastromyzon is as follows:

AkeytoPseudogastromyzon

1(2) Top of the second dermal ridge not exceeding the

fourth, fourth dermal ridge generally ﬂat; side of lat-

eral body with irregular, coarse horizontal bands and

blotches dispersed along the lateral line . . P. myersi

2(1) Top of the second dermal ridge exceeding the

fourth, fourth dermal ridge distinctively acclivous;

side of lateral body with vertical stripes, or irregular

circular spots, or black dots, or is pattern-less

3(4) Second dermal ridge bimodal . ......... P. laticeps

4(3) Second dermal ridge unimodal

5(6) Sides of body with irregular circular spots . P. cheni

6(5) Sides of body with vertical stripes ..... P. fasciatus

Estimating species divergence time and ancestral

range reconstruction analysis

We have ascertained that the genera Labigastromy-

zon and Pseudogastromyzon together constitute a mono-

phyletic clade, while Erromyzon displays a sister-group

relationship with this clade. As illustrated in Fig. 9II,

species belonging to the genus Erromyzon exhibit a

rudimentary dermal ridge structure on their lower lip. Al-

though lacking a sophisticated adhesive apparatus, this

characteristic implies a relatively proximate genetic rela-

tionship with the other two genera.

The ancestral range reconstruction analysis infers that

the divergence of Erromyzon and Labigastromyzon,the

divergence of Pseudogastromyzon and Labigastromyzon,

and the internal diversiﬁcation of Labigastromyzon took

place within areas A or B, with population dispersal driv-

ing the diversiﬁcation process. Concurrently, diversiﬁca-

tion within the genus Pseudogastromyzon transpired in

area B, where both dispersal and vicariance events con-

tributed to the divergence between P. fasciatus and P.

cheni.

Drawing upon these ﬁndings, we deduce that the

overarching trend of expansion and diversiﬁcation for

these three taxonomic groups progressed from west to

east and from south to north. At the close of the Miocene

epoch (approximately 6.05 Mya), the Himalayan tectonic

period culminated, giving rise to numerous rivers in

southern China adopting a west-to-east ﬂow direction.

This shift emerged as one of the propelling forces for the

dispersal of Pseudogastromyzon and Labigastromyzon

ancestors. In the early Pliocene epoch (approximately

5.55 Mya), Labigastromyzon and Pseudogastromyzon ex-

perienced further divergence in the southeastern coastal

region of Guangdong, characterized by a more intricate

adhesive apparatus and fusion of the outer margins of

the lower and upper lips. This adaptation conferred

enhanced adhesion, beneﬁcial for navigating the more

tumultuous streams resulting from monsoonal inﬂuence

and heightened precipitation. This adaptation facilitated

the further dispersal of Pseudogastromyzon in the coastal

region, engendering new species during its northward

expansion. Alterations in ﬂow direction due to mountain

erosion likely catalyzed this process, and this northward

dispersal signiﬁes a historical northeastward ﬂowing

stage of the river systems along the Guangdong–Fujian

coast.

In accordance with species distribution surveys, we

have observed that, in contrast to the majority of fresh-

water ﬁsh, these species tend to populate both sides of

the watershed. This distribution pattern is attributable to

their specialized body structures, which have bolstered

their capacity for upstream dispersal within river basins.

We contend that the dispersal of these ﬁsh species is

contingent upon mountainous terrain, and they rely on

the shifting direction of mountain streams to disseminate

along the slopes.

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. 19

J. Ch en et al.

Figure 9 (I) Results of the divergence time and ancestral range reconstruction analysis, where the values on the nodes represent mean

ages (above) and the 95% HPD intervals of the divergence time estimates (below). The circular symbols on the nodes indicate the

geographical region. (II) The lower lip of Erromyzon sinensis exhibits a very primitive ridge-like structure, but has not yet developed

an adhesive apparatus. (III) Regional classiﬁcation based on species distribution.

ACKNOWLEDGMENTS

We would like to express our profound gratitude to Ms.

Jiajia Li for her invaluable assistance in the meticulous

measurement and illustration of specimens. Additionally,

we extend our heartfelt appreciation to Mr. Jiajun Zhou,

Mr. Zhenguan Zhao, Mr. Qianyu Liang, Mr. Zhiqi Xiao,

and Mr. Chuan Yao for their assiduous efforts in collect-

ing specimens from Zhejiang, Fujian, Guangxi, Hunan,

and Jiangxi provinces, respectively. Furthermore, we are

grateful to Mr. Zhongwu Chi and Mr. Xinrui Pu for their

expert guidance and assistance in conducting species sur-

veys within Yunnan province, as well as Mr. Zhaoning

Peng for his contribution to the species survey in Hainan

province. This work was supported by the National Nat-

ural Science Foundation of China (NSFC) (Grant 31093

430) and the National Special Program of Basic Research

Works for Science and Technology (2015FY110200).

CONFLICT OF INTEREST

The authors declare that they have no competing inter-

ests.

REFERENCES

Bankevich A, Nurk S, Antipov D et al. (2012). SPAdes: a

new genome assembly algorithm and its applications to

single-cell sequencing. Journal of Computational Biol-

ogy 19, 455–77.

Boulenger GA (1894). Descriptions of a new lizard and a

new ﬁsh obtained in Formosa by Mr. Holst. Journal of

Natural History 14, 462–63.

Chen YX, Chen YS, Shi CM et al. (2018). SOAP-

nuke: A MapReduce acceleration-supported software

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

Phylogeny of Pseudogastromyzon

for integrated quality control and preprocessing of

high-throughput sequencing data. GigaScience 7,

gix120.

Chen YY (1980). Systematic studies on the ﬁshes of the

family Homalopteridae of China II. Classiﬁcation of

the ﬁshes of the subfamily Gastromyzoninae. Acta Hy-

drobiologica Sinica 7, 95–120.

Chen YY, Tang WQ (2000). Homalopteridae. In: Le PQ

et al., ed. Fauna Sinica, Osteichthyes, Cypriniformes

III. Science Press, Beijing, pp. 503–19.

Donath A, Jühling F, Al-Arab M et al. (2019). Im-

proved annotation of protein-coding genes boundaries

in metazoan mitochondrial genomes. Nucleic Acids

Research 47, 10543–52.

Fang PW (1930). New and inadequately known Homa-

lopterine Loaches of China. Contributions from the Bi-

ological Laboratory of the Science Society of China.

(Zoological Series) 6, 24–43.

Fang PW (1933). Notes on some Chinese Homalopterid

loaches. Sinensia 4, 39–50.

Froese R, Pauly D (2010). FishBase: Fisheries Cen-

tre. University of British Columbia Vancouver, British

Columbia.

He SL, Zhang JP, Xu MJ (2000). New Records of Fish

and Fish Fauna in Hunan Province. Journal of Hu-

nan Agricultural University (Natural Sciences) 26,

379–82.

Helfrich P, Rieb E, Abrami G, Lücking A, Mehler A

(2018). TreeAnnotator: Versatile visual annotation of

hierarchical text relations. Proceedings of the Eleventh

International Conference on Language Resources and

Evaluation (LREC 2018). European Language Re-

sources Association, France.

Hoang DT, Chernomor O, Von Haeseler A, Minh BQ,

Vinh LS (2018). UFBoot2: Improving the ultrafast

bootstrap approximation. Molecular Biology and Evo-

lution 35, 518–22.

Hora SL (1931). Classiﬁcation of the Homalopterid

ﬁshes. Records of the Zoological Survey of India 33,

67–69.

Hora SL (1932). Classiﬁcation, bionomics and evolution

of Homalopterid ﬁshes. Memoirs of the Indian Museum

12, 261–330.

Huang ZJ, He JG, Tang JJ, Xu XP, Zheng JQ (2009).

Germplasm resource investigation of wild freshwater

ﬁsh in Beijiang River of Guangdong. Chinese Journal

of Ecology 28, 1489–93.

Kalyaanamoorthy S, Minh BQ, Wong TKF, Von Haeseler

A, Jermiin LS (2017). ModelFinder: Fast model selec-

tion for accurate phylogenetic estimates. Nature Meth-

ods 14, 587–89.

Kottelat M (2001). Freshwater Fishes of Northern Viet-

nam: A Preliminary Check-list of the Fishes known

or Expected to Occur in Northern Vietnam with Com-

ments on Systematics and Nomenclature. World Bank,

Washington, DC.

Kottelat M (2012). Conspectus Cobitidum: An Inventory

of the Loaches of the World (Teleostei: Cypriniformes:

Cobitoidei). Rafﬂes Bulletin of Zoology. National Uni-

versity of Singapore Press, Singapore.

Kottelat M (2013). The Fishes of the Inland Waters of

Southeast Asia: A Catalogue and Core Bibliography of

the Fishes known to Occur in Freshwaters, Mangroves

and Estuaries. Rafﬂes Bulletin of Zoology. National

University of Singapore, Singapore.

Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott

B (2017). PartitionFinder 2: New methods for selecting

partitioned models of evolution for molecular and mor-

phological phylogenetic analyses. Molecular Biology

and Evolution 34, 772–73.

Li HJ, Lin XT (2010). Preliminary study on ﬁsh commu-

nities in mountain forest streams in Western Guang-

dong. Jiangsu Agricultural Sciences 5, 495–504.

Li HJ, Xie CX (2004). Funna composition and resources

protection of freshwater ﬁsh in forest streams in the

central north of Guangxi Province. Journal of North-

east Forestry University 32, 89–93.

Li J, Luo JR, Li XH, Tan XC, Wang C, Guo SC (2007).

Investigation of ﬁsh resources and analysis of resources

decline along Lianjiang River. Freshwater Fisheries

37, 49–53.

Li SQ (1998). Study on the homalopterid ﬁshes from Fu-

jian Province. Journal of Fisheries of China 22, 260–

64.

Liang YS (1942). Notes on some species of homalopterid

loaches referring to Pseudogastromyzon from Fukien.

Contributions from the Research Institute of Zoology

and Botany, Fukien Provincial Academy 1, 1–10.

Liu XJ, Qin JJ, Ao XF et al. (2020). Species diversity

of ﬁsh in the Luoxiao Mountains region. Biodiversity

Science 28, 889.

Mai D (1978). Identiﬁcation of the Fresh-Water Fishes of

North Vietnam. Scientiﬁc & Technology Publisher, Ha

Noi.

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd. 21

J. Ch en et al.

Matzke NJ (2013). BioGeoBEARS: BioGeography with

Bayesian (and likelihood) evolutionary analysis in R

Scripts. R package, version 0.2 1, 2013.

Nguyen LT, Schmidt HA, Von Haeseler A, Minh BQ

(2015). IQ-TREE: A fast and effective stochastic algo-

rithm for estimating maximum-likelihood phylogenies.

Molecular Biology and Evolution 32, 268–74.

Nichols JT (1925). A new homalopterin loach from

Fukien. American Museum Novitates (167), 1–2.

Nichols JT (1931). Crossostoma fangi, a new loach from

near Canton, China. Lingnan Science Journal 10, 263–

64.

Pan JH, Liu CH, Zheng WB (1984). Study on the ﬁsh

fauna of the Beijiang River, Guangdong Province.

Journal of South China Normal University (Natural

Science Edition) 1, 27–40.

Rambaut A (2018). FigTree v1.4.4. Available from URL:

http://tree.bio.ed.ac.uk/software/ﬁgtree/

Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA

(2018). Posterior summarization in Bayesian phyloge-

netics using Tracer 1.7. Systematic Biology 67, 901–4.

Ronquist F, Huelsenbeck JP (2003). MrBayes 3: Bayesian

phylogenetic inference under mixed models. Bioinfor-

matics 19, 1572–74.

Sauvage HE (1878). Note sur quelques Cyprinidae et Co-

bitidae d’espèces inédites, provenant des eaux douces

de la Chine. Bulletin de la Société philomathique de

Paris (7th Série) 2, 86–90.

Song BL (1992). Preliminary investigation on ﬁshes in

Jianjiang River. Journal of Zhanjiang Fisheiries Col-

lege 12, 83–86.

Suchard MA, Lemey P, Baele G, Ayres DL, Drummond

AJ, Rambaut A (2018). Bayesian phylogenetic and

phylodynamic data integration using BEAST 1.10.

Virus Evolution 4, vey016.

Tamura K, Stecher G, Kumar S (2021). MEGA11: Molec-

ular evolutionary genetics analysis version 11. Molec-

ular Biology and Evolution 38, 3022–27.

Tang WQ, Chen YY (1996). Ultrastructural observation

on the chin adhesive apparatus and subgenus division

of Pseudogastromyzon.Acta Zoologica Sinica 42, 231–

36.

Xia X, Xie Z (2001). DAMBE: software package for data

analysis in molecular biology and evolution. Journal of

Heredity 92, 371–73.

Xiao JL, Deng WD, Peng HY, Qiu HW, Li RS. (2021).

Preliminary analysis of ﬁsh biodiversity in Guang-

dong Shixing Nanshan Provincial Nature Reserve.

Journal of Shaoguan University·Natural Science 42,

55–60.

Xiao WH, Zhang YP (2000). Genetics and evolution

of mitochondrial DNA in ﬁsh. Acta Hydrobiologica

Sinica 24, 384–91.

Xu MY, Guo LD, Gu SQ et al. (2020). TGS-GapCloser:

a fast and accurate gap closer for large genomes with

low coverage of error-prone long reads. GigaScience 9,

giaa094.

Yu Y, Harris AJ, Blair C, He X (2015). RASP (Re-

construct Ancestral State in Phylogenies): A tool for

historical biogeography. Molecular Phylogenetics and

Evolution 87, 46–49.

Yuan SB, Liu SQ, Xiang Y et al. (2020). Current status

and protection countermeasures of ﬁsh fauna structure

of Centian River National Wetland Park in Jianghua,

Hunan. Biotic Resources 42, 644–51.

Zhang D, Gao FL, Jakovli´

cL,ZouHet al. (2020). Phylo-

Suite: An integrated and scalable desktop platform for

streamlined molecular sequence data management and

evolutionary phylogenetics studies. Molecular Ecology

Resources 20, 348–55.

Zheng CY, Chen YY (1980). The homalopterid ﬁshes

from Guangdong Province, China. Acta Zootaxonom-

ica Sinica 5, 89–101.

Zheng CY, Li JP (1986). A preliminary review

of the genus Pseudogastromyzon (Homalopteridae,

Cyprinidae) of China. Journal of Science and Medicine

of Jinan University 1, 75–84.

Zheng CY (1981). The homalopterid ﬁshes from Guang-

dong province, China (continuity). Journal of Jinan

University (Natural Science & Medicine Edition) 1,

55–63.

Cite this article as:

Chen J, Chen Y, Tang W, Lei H, Yang J, Song X (2023). Resolving phylogenetic relationships and taxonomic revision

in the Pseudogastromyzon (Cypriniformes, Gastromyzonidae) genus: molecular and morphological evidence for a

new genus, Labigastromyzon.Integrative Zoology 00 1–22. https://doi.org/10.1111/1749-4877.12761

Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.

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Mar 2024
Mitt Mus Natur Be Zool Reihe

Bashimyzon, new genus, is here established for Erromyzon damingshanensis, and a new species of the genus is described from the You-Jiang of the Pearl River (=Zhu-Jiang in mandarin Chinese) basin in Guangxi Province, South China. This new genus has a small gill opening above the pectoral-fin base and short pectoral fins extending backwards short of pelvic-fin insertions, both characters combined to separate it from all currently-recognized gastromyzontid genera except Erromyzon and Protomyzon, but differs from the two genera in having a larger gap between the posterior edge of eye and the vertical through the pectoral-fin insertion and very small fleshy lobes posterior to the maxillary-barbel bases. It is further distinct from its most similar genus Erromyzon in having a relatively larger gill opening, fewer branched pectoral-fin rays folded against body, and more posteriorly placed pectoral fins with a shorter fin base. Bashimyzon cheni, new species, and B. damingshanensis, the single congeneric species, differ in number of lateral-line pored scales, body coloration, and cephalic contour, and also in substantial genetic divergence.

Taxonomic resolution of the hillstream suck-loach Beaufortia pingi species group (Cypriniformes, Gastromyzontidae) and two new species from Southwest China– Beaufortia granulopinna and Beaufortia viridis

Article

Full-text available

Jul 2024

Two new species, Beaufortia granulopinna and Beaufortia viridis , are described from the upper Pearl River system in southwest China. Both species share the characteristics of the Beaufortia pingi species group, including prominent vertical stripes on the lateral body and pinnate-type lower lips, distinguishing them from other Beaufortia species. Beaufortia granulopinna is differentiated from other species in the group by possessing a unique set of characteristics: the presence of well-developed prominent tubercles on the first 6–9 pectoral fin rays in adults; and a significant proportion (54.76%) of individuals experiencing blurriness or absence of vertical stripes in the mid-section of the lateral body upon reaching adulthood. The minimum interspecific genetic distance within the genus based on mitochondrial cyt b gene sequences is 10.80%. Beaufortia viridis is distinguished from other species in the group by consistently exhibiting vertical stripes of uniform length, width, and spacing across all stages of growth; the absence of tubercles on the branched rays of pectoral fins; and a body coloration of dark cyan to green. The minimum interspecific genetic distance within the genus based on mitochondrial cyt b gene sequences is 4.60%. Molecular phylogenetic results confirm that the Beaufortia pingi species group forms a monophyletic clade, which is congruent with morphological classification findings. This study also addresses and resolves the taxonomic ambiguity surrounding Beaufortia pingi and Beaufortia zebroida , providing a redescription of these taxa.

MEGA11: Molecular Evolutionary Genetics Analysis Version 11

Article

Full-text available

Apr 2021
MOL BIOL EVOL

The Molecular Evolutionary Genetics Analysis (MEGA) software has matured to contain a large collection of methods and tools of computational molecular evolution. Here, we describe new additions that make MEGA a more comprehensive tool for building timetrees of species, pathogens, and gene families using rapid relaxed-clock methods. Methods for estimating divergence times and confidence intervals are implemented to use probability densities for calibration constraints for node-dating and sequence sampling dates for tip-dating analyses, which will be supported by new options for tagging sequences with spatiotemporal sampling information, an expanded interactive Node Calibrations Editor, and an extended Tree Explorer to display timetrees. We have now added a Bayesian method for estimating neutral evolutionary probabilities of alleles in a species using multispecies sequence alignments and a machine learning method to test for the autocorrelation of evolutionary rates in phylogenies. The computer memory requirements for the maximum likelihood analysis are reduced significantly through reprogramming, and the graphical user interface (GUI) has been made more responsive and interactive for very big datasets. These enhancements will improve the user experience, quality of results, and the pace of biological discovery. Natively compiled GUI and command-line versions of MEGA11 are available for Microsoft Windows, Linux, and macOS from www.megasoftware.net.

TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads

Article

Full-text available

Sep 2020

Background: Analyses that use genome assemblies are critically affected by the contiguity, completeness, and accuracy of those assemblies. In recent years single-molecule sequencing techniques generating long-read information have become available and enabled substantial improvement in contig length and genome completeness, especially for large genomes (>100 Mb), although bioinformatic tools for these applications are still limited. Findings: We developed a software tool to close sequence gaps in genome assemblies, TGS-GapCloser, that uses low-depth (∼10×) long single-molecule reads. The algorithm extracts reads that bridge gap regions between 2 contigs within a scaffold, error corrects only the candidate reads, and assigns the best sequence data to each gap. As a demonstration, we used TGS-GapCloser to improve the scaftig NG50 value of 3 human genome assemblies by 24-fold on average with only ∼10× coverage of Oxford Nanopore or Pacific Biosciences reads, covering with sequence data up to 94.8% gaps with 97.7% positive predictive value. These improved assemblies achieve 99.998% (Q46) single-base accuracy with final inserted sequences having 99.97% (Q35) accuracy, despite the high raw error rate of single-molecule reads, enabling high-quality downstream analyses, including up to a 31-fold increase in the scaftig NGA50 and up to 13.1% more complete BUSCO genes. Additionally, we show that even in ultra-large genome assemblies, such as the ginkgo (∼12 Gb), TGS-GapCloser can cover 71.6% of gaps with sequence data. Conclusions: TGS-GapCloser can close gaps in large genome assemblies using raw long reads quickly and cost-effectively. The final assemblies generated by TGS-GapCloser have improved contiguity and completeness while maintaining high accuracy. The software is available at https://github.com/BGI-Qingdao/TGS-GapCloser.

PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies

Article

Full-text available

Jan 2020
MOL ECOL RESOUR

Multigene and genomic data sets have become commonplace in the field of phylogenetics, but many existing tools are not designed for such data sets, which often makes the analysis time‐consuming and tedious. Here, we present PhyloSuite, a (crossplatform, open‐source, stand‐alone Python graphical user interface) user‐friendly workflow desktop platform dedicated to streamlining molecular sequence data management and evolutionary phylogenetics studies. It uses a plugin‐based system that integrates several phylogenetic and bioinformatic tools, thereby streamlining the entire procedure, from data acquisition to phylogenetic tree annotation (in combination with iTOL). It has the following features: (a) point‐and‐click and drag‐and‐drop graphical user interface; (b) a workplace to manage and organize molecular sequence data and results of analyses; (c) GenBank entry extraction and comparative statistics; and (d) a phylogenetic workflow with batch processing capability, comprising sequence alignment (mafft and macse), alignment optimization (trimAl, HmmCleaner and Gblocks), data set concatenation, best partitioning scheme and best evolutionary model selection (PartitionFinder and modelfinder), and phylogenetic inference (MrBayes and iq‐tree). PhyloSuite is designed for both beginners and experienced researchers, allowing the former to quick‐start their way into phylogenetic analysis, and the latter to conduct, store and manage their work in a streamlined way, and spend more time investigating scientific questions instead of wasting it on transferring files from one software program to another.

Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes

Article

Full-text available

Oct 2019
NUCLEIC ACIDS RES

With the rapid increase of sequenced metazoan mitochondrial genomes, a detailed manual annotation is becoming more and more infeasible. While it is easy to identify the approximate location of protein-coding genes within mitogenomes, the peculiar processing of mitochondrial transcripts, however, makes the determination of precise gene boundaries a surprisingly difficult problem. We have analyzed the properties of annotated start and stop codon positions in detail, and use the inferred patterns to devise a new method for predicting gene boundaries in de novo annotations. Our method benefits from empirically observed prevalances of start/stop codons and gene lengths, and considers the dependence of these features on variations of genetic codes. Albeit not being perfect, our new approach yields a drastic improvement in the accuracy of gene boundaries and upgrades the mitochondrial genome annotation server MITOS to an even more sophisticated tool for fully automatic annotation of metazoan mitochondrial genomes.

Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10

Article

Full-text available

Jun 2018

The Bayesian Evolutionary Analysis by Sampling Trees (BEAST) software package has become a primary tool for Bayesian phylogenetic and phylodynamic inference from genetic sequence data. BEAST unifies molecular phylogenetic reconstruction with complex discrete and continuous trait evolution, divergence-time dating, and coalescent demographic models in an efficient statistical inference engine using Markov chain Monte Carlo integration. A convenient, cross-platform, graphical user interface allows the flexible construction of complex evolutionary analyses.

Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7

Article

Full-text available

Apr 2018
SYST BIOL

Bayesian inference of phylogeny using Markov chain Monte Carlo (MCMC) (Drummond et al., 2002; Mau et al., 1999; Rannala and Yang, 1996) flourishes as a popular approach to uncover the evolutionary relationships among taxa, such as genes, genomes, individuals or species. MCMC approaches generate samples of model parameter values - including the phylogenetic tree -drawn from their posterior distribution given molecular sequence data and a selection of evolutionary models. Visualising, tabulating and marginalising these samples is critical for approximating the posterior quantities of interest that one reports as the outcome of a Bayesian phylogenetic analysis. To facilitate this task, we have developed the Tracer (version 1.7) software package to process MCMC trace files containing parameter samples and to interactively explore the high-dimensional posterior distribution. Tracer works automatically with sample output from BEAST (Drummond et al., 2012), BEAST2 (Bouckaert et al., 2014), LAMARC (Kuhner, 2006), Migrate (Beerli, 2006), MrBayes (Ronquist et al., 2012), RevBayes (Höhna et al., 2016) and possibly other MCMC programs from other domains.

TreeAnnotator: Versatile Visual Annotation of Hierarchical Text Relations

Conference Paper

Full-text available

May 2018

We introduce TreeAnnotator, a graphical tool for annotating tree-like structures, in particular structures that jointly map dependency relations and inclusion hierarchies, as used by Rhetorical Structure Theory (RST). TreeAnnotator is browser-based, embedded within the UIMA framework and provides two visualization modes. TreeAnnotator’s interoperability exceeds similar tools, providing a wider range of formats, while annotation work can be completed more quickly due to a revised input method for RST dependency relations. TreeAnnotator offers a multiple window view, which allows users to inspect several annotations side by side. For storing and versioning annotations, the UIMA Database Interface (UIMA DI) was developed to save documents based on a pre-defined type system. These features not only connect TreeAnnotator annotations to modern technological and dialog theoretical work, but set it apart from related tools. The ease of use of TreeAnnotator and its newly designed user interface is evaluated in a user study consisting of annotating rhetorical relations with TreeAnnotator and the classic RSTTool.

SOAPnuke: A MapReduce Acceleration supported Software for integrated Quality Control and Preprocessing of High-Throughput Sequencing Data

Article

Full-text available

Dec 2017

Quality Control (QC) and preprocessing are essential steps for sequencing data analysis to ensure the accuracy of results. However, existing tools cannot provide a satisfying solution with integrated comprehensive functions, proper architectures and highly-scalable acceleration. In this article, we demonstrate SOAPnuke as a tool with abundant functions for a “QC-Preprocess-QC” workflow and MapReduce acceleration framework. Four modules with different preprocessing functions are designed for processing datasets from genomic, small RNA (sRNA), Digital Gene Expression (DGE) and metagenomic experiments respectively. As a workflow-like tool, SOAPnuke centralizes processing functions in one executable and predefine their order to avoid the necessity of reformatting different files when switching tools. Furthermore, the MapReduce framework enables large scalability to distribute all the processing works to an entire compute cluster. We conducted a benchmarking where SOAPnuke and other tools are used to preprocess ∼30x NA12878 dataset published by GIAB. The standalone operation of SOAPnuke struck a balance between resource occupancy and performance. When accelerated on 16 working nodes with MapReduce, SOAPnuke achieved ∼5.7 times of the fastest speed of other tools.

UFBoot2: Improving the Ultrafast Bootstrap Approximation

Article

Full-text available

Oct 2017

The standard bootstrap (SBS), despite being computationally intensive, is widely used in maximum likelihood phylogenetic analyses. We recently proposed the ultrafast bootstrap approximation (UFBoot) to reduce computing time while achieving more unbiased branch supports than SBS under mild model violations. UFBoot has been steadily adopted as an efficient alternative to SBS and other bootstrap approaches. Here, we present UFBoot2, which substantially accelerates UFBoot and reduces the risk of overestimating branch supports due to polytomies or severe model violations. Additionally, UFBoot2 provides suitable bootstrap resampling strategies for phylogenomic data. UFBoot2 is 778 times (median) faster than SBS and 8.4 times (median) faster than RAxML rapid bootstrap on tested datasets. UFBoot2 is implemented in the IQ-TREE software package version 1.6 and freely available at http://www.iqtree.org.

SYSTEMATIC STUDIES ON THE FISHES OF THE FAMILY HOMALOPTERIDAE OF CHINA Ⅱ.CLASSIFICATION OF THE FISHES OF THE SUBFAMILY GASTROMYZONINAE

Article

Jan 1980

Yiyu Chen

Resolving phylogenetic relationships and taxonomic revision in the Pseudogastromyzon (Cypriniformes, Gastromyzonidae) genus: molecular and morphological evidence for a new genus, Labigastromyzon

Abstract and Figures

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