diversity
Article
Review of the Rheotanytarsus muscicola Species Group from
China (Diptera: Chironomidae) †
Xin Qi 1,2 , Yuan Yao 3 , Wen-Bin Liu 3 , Chun-Cai Yan 3 , Xin-Hua Wang 4 and Xiao-Long Lin 1,4, *
Citation: Qi, X.; Yao, Y.; Liu, W.-B.;
Yan, C.-C.; Wang, X.-H.; Lin, X.-L.
Review of the Rheotanytarsus
muscicola Species Group from China
(Diptera: Chironomidae). Diversity
2022, 14, 383. https://doi.org/
10.3390/d14050383
Academic Editor: Ming Bai
Received: 9 April 2022
Accepted: 10 May 2022
Published: 12 May 2022
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Diversity 2022, 14, 383. https://doi.org/10.3390/d14050383
1 Joint Laboratory of Xianju National Park & Taizhou University, Taizhou University, Taizhou 318000, China;
qixin0612@tzc.edu.cn
2 College of Life Sciences, Taizhou University, Taizhou 318000, China
3 Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity, Tianjin Normal University,
Tianjin 300387, China; yuanyao.work@hotmail.com (Y.Y.); skylwb@tjnu.edu.cn (W.-B.L.);
skyycc@tjnu.edu.cn (C.-C.Y.)
4 College of Life Sciences, Nankai University, Tianjin 300071, China; xhwang@nankai.edu.cn
* Correspondence: lin880224@gmail.com
† urn:lsid:zoobank.org:act:049C6E91-FA03-4FA4-9C5A-49BA0AF478ED.
Abstract: The Rheotanytarsus muscicola species group is generally considered to be a taxonomically
difficult group of non-biting midges (Diptera: Chironomidae). In this study, we review the R. mus-
cicola species group based on the adult males from China using morphology and DNA barcodes.
Rheotanytarsus ferringtoni Lin & Yao sp. n. is described and figured, and four species (Rheotanytar-
sus falcipedius Kyerematen, Andersen & Sæther, 2000; Rheotanytarsus fluminis Kawai & Sasa, 1985;
Rheotanytarsus illiesi Siebert, 1979; and Rheotanytarsus photophilus Goetghebuer, 1921) new to China
are redescribed, figured and discussed. An updated key to known adult males of the R. muscicola
species group is presented. Our study shows concordance between morphological species concepts
and DNA barcodes.
Keywords: Tanytarsini; identification key; COI; new species; China
1. Introduction
Rheotanytarsus Thienemann & Bause, 1913 (Diptera: Chironomidae) is one of the most
species-rich genera of the tribe Tanytarsini in the subfamily Chironominae, containing more
than 100 described species worldwide [1–12]. Larvae of Rheotanytarsus are rheobiontic,
occurring in large rivers and the littoral of lakes [13]. Rheotanytarsus has been revised
and divided into 15 species groups over the last few decades [9]. According to a cladistic
analysis using morphological data by Sæther & Kyerematen in 2001 [9], the Rheotanytarsus
muscicola species group was erected, including 15 species in Afrotropical, Oriental and
Holarctic regions [6,14]. Prior to this study, two species of the Rheotanytarsus muscicola
species group were recorded in China [2,5].
Over the last decades, the DNA barcode corresponding to the 658 bp fragment of
the mitochondrial gene cytochrome c oxidase I (COI) has been identified as the core of a
global bio-identification system at the species level [15,16] and has proven to be useful in
the delimitation of non-biting midge species and provided important evidence to confirm
new species [17–28]. The morphological similarity between species within the R. muscicola
species group has likely led to misidentifications and underestimation of species diversity
in the past. In general, DNA barcode data and morphology can give a good estimation in
closely related species.
In this study, we review species within the Rheotanytarsus muscicola species group (Figure 1)
from China, diagnosing and describing them based on morphology and DNA barcodes. An
updated key to known adult males of the R. muscicola species group is presented.
https://www.mdpi.com/journal/diversity
Diversity 2022, 14, 383 2 of 16
Diversity 2022, 14, x FOR PEER REVIEW 2 of 17

Figure 1. Adult males of Rheotanytarsus muscicola species group. (a) Rheotanytarsus falcipedius, dorsal
Figure 1. Adult males
view; of Rheotanytarsus
(b) Rheotanytarsus muscicola
ferringtoni species
sp. n., lateral group.
view; (a) Rheotanytarsus
(c) Rheotanytarsus falcipedius,
fluminis, lateral view; (d)dorsal
view; (b) Rheotanytarsus
Rheotanytarsusferringtoni
illiesi, lateral sp.
view;n.,
(e) Rheotanytarsus (c) Rheotanytarsus
lateral view;muscicola, ventral view; (f)fluminis, lateral
Rheotanytarsus pho- view;
tophilus, dorsal view.
(d) Rheotanytarsus illiesi, lateral view; (e) Rheotanytarsus muscicola, ventral view; (f) Rheotanytarsus
photophilus, dorsal view. and Methods
2. Materials
2.1. Taxon Sampling and Identification
2. Materials and Methods
A total of 16 specimens of the Rheotanytarsus muscicola species group were collected
2.1. Taxon Sampling and Identification
from China. Adult males were collected by using malaise trap and light trap. The material
A total of 16 specimens
examined 85%Rheotanytarsus
was storedofinthe ethanol and mountedmuscicola
on slidesspecies
followinggroup were spec-
the procedure collected
ified in males
from China. Adult [2,10,29,30].
were Specimens
collectedwere
byidentified using taxonomic
using malaise trap and revisions and species
light trap. de-
The material
scriptions [3].The morphological nomenclature follows Sæther [31]. Color is described as
examined was stored in 85% ethanol and mounted on slides following the procedure
observed in specimens mounted in Euparal on microscopy slides. Digital photographs of
specified in [2,10,29,30].
slide-mountedSpecimens
specimens werewere identified
taken using
with 300 dpi taxonomic
resolution using a revisions and
Nikon Digital species
Sight
descriptions [3]. The
DS-Fil morphological
camera nomenclature
mounted on Nikon follows Sæther
Eclipse 80i compound [31].
microscope Color
using is described
the software
as observed inNIS-Elements
specimensF mounted
v.4.60.00. The
in voucher
Euparal specimens were deposited
on microscopy at Digital
slides. the College of Life
photographs
Sciences, Nankai University, Tianjin, China (NKU).
of slide-mounted specimens were taken with 300 dpi resolution using a Nikon Digital Sight
DS-Fil camera 2.2.mounted on Nikon
Molecular Laboratory Eclipse 80i compound microscope using the software
Work
NIS-Elements F v.4.60.00. The voucher specimens
Before slide preparation, genomic DNA of were deposited
most specimens wasatextracted
the College of Life
from the
Sciences, Nankai University, Tianjin, China (NKU).
head-thorax using Qiagen DNA Blood and Tissue Kit according to the manufacture’s in-
struction. PCR amplifications of COI barcodes with the universal primers LCO1490 and
2.2. MolecularHCO2198
Laboratory
[32] Work
were performed following the protocol from Lin et al. [18]. Sanger sequenc-
ing of the purified PCR products was carried out on the ABI 3730 at the BGI (Beijing,
Before slide preparation, genomic DNA of most specimens was extracted from the head-
China). In addition, genomic DNA extraction from three legs, PCR amplification, and
thorax using high-throughput
Qiagen DNA Blood andofTissue
sequencing Kit according
the specimens to theatmanufacture’s
were conducted instruction.
the Canadian Centre for
PCR amplifications of COI barcodes
DNA Barcoding with the
(CCDB, University of universal primers
Guelph, Canada) usingLCO1490 and HCO2198 [32]
standard high-throughput
were performed protocols [33]. DNA
following the samples
protocol arefrom
deposited
Lin etat al.
the [18].
College of Lifesequencing
Sanger Sciences, Nankai Univer-
of the purified
sity, Tianjin, China, and the CCDB.
PCR products was carried out on the ABI 3730 at the BGI (Beijing, China). In addition, genomic
DNA extraction fromBarcode
2.3. DNA three Analysis
legs, PCR amplification, and high-throughput sequencing of the
specimens were conducted
Raw sequencesat the Canadian
were edited andCentre for in
assembled DNA Barcoding
Geneious (CCDB,
Prime version University
2021.0.3 and of
Guelph, Canada) using
aligned usingstandard
MUSCLEhigh-throughput
[34] implemented in protocols
MEGA 11.0 [33]. DNA
[35] samples
to check are deposited
stop codons. To
at the College of Life Sciences, Nankai University, Tianjin, China, and the CCDB.

2.3. DNA Barcode Analysis

Raw sequences were edited and assembled in Geneious Prime version 2021.0.3 and aligned
using MUSCLE [34] implemented in MEGA 11.0 [35] to check stop codons. To obtain DNA
barcodes, we searched for public COI barcodes of the Rheotanytarsus muscicola species group
that were longer than 500 base pairs with a lack of stop codons in the Barcode of Life Data
System (BOLD, http://www.boldsystems.org/ (accessed on 30 March 2022)) [36]. In total, a
dataset entitled “DNA barcodes of Rheotanytarsus muscicola species group (DS-MUSCICO)”,
including 22 COI barcodes of Rheotanytarsus muscicola species group, was generated on BOLD
Diversity 2022, 14, 383 3 of 16

(30 March 2022), with 16 COI barcodes representing six species originating from this study, while
the single COI barcode of Rheotanytarsus guanacastensis Kyerematen and Andersen 2002 was
publicly acquired from BOLD. All COI sequences were applied to the Barcode Index Number
(BIN) system on BOLD [37]. In addition, a neighbor-joining (NJ) [38] tree was constructed based
on the 22 COI barcodes using the Kimura 2-Parameter (K2P) model [39] with 1000 bootstrap
replicates and pairwise deletion in MEGA.

3. Results and Discussion

3.1. DNA Barcode Analysis
The aligned 22 COI sequences ranged from 589 to 658 base pairs, including 12 se-
quences with a full barcode length of 658 base pairs. These 22 sequences were assigned to
eight BINs, including four new BINs. The NJ tree (Figure 2) based on COI DNA barcodes
of the Rheotanytarsus muscicola species group revealed seven well-separated clusters repre-
senting seven morphospecies. The new species Rheotanytarsus ferringtoni sp. n. separated
from R. guanacastensis by more than 12% divergence in COI barcodes. Interestingly, COI se-
quences of R. muscicola were grouped into three different clades. Unfortunately, we had no
access to examine the vouchers of R. muscicola from Finland (BIN: BOLD:AAW4702) and Xin-
jiang, China (BIN: BOLD:ADG9631). Since R. illiesi keys to the same BIN (BOLD:ADG9631),
we could confirm that the larva named as “R. muscicola” (BOLD Sample ID: Rheotany-
tarsus muscicola Habahe) from Xinjiang was misidentified. The BIN (BOLD:AAW4702)
included four individuals from Finland (BOLD Sample ID: ZMUO.024880, ZMUO.024881,
ZMUO.025104 and ZMUO.025105), with 7% genetic distance to the R. photophilus. Although
we didn’t examine the vouchers from Finland, we believe that R. photophilus includes two
BINs resulting from deep intraspecific divergence. In general, the average intraspecific
divergence in COI p-distance (4–6%) is slightly higher than other insects, and the maximum
Diversity 2022, 14,intraspecific divergence can be up to 10% [18].
x FOR PEER REVIEW 4 of 17

Figure 2. Neighbor-joining tree for seven species of the Rheotanytarsus muscicola species group based
Figure 2. Neighbor-joining tree for seven species of the Rheotanytarsus muscicola species group based
on K2P distance in DNA barcodes. Numbers on branches represent bootstrap support (>70%) based
on K2P distance in DNA
on 1000 barcodes.
replicates; Numbers
scale equals on branches
K2P genetic distance. represent bootstrap support (>70%) based
on 1000 replicates; scale equals K2P genetic distance.
3.2. Taxonomy
3.2. Taxonomy Adult males of the species in the Rheotanytarsus muscicola species group have the fol-
lowing morphological characters: the anal tergite bands developed and of V-type, and the
Adult males of the species in the Rheotanytarsus muscicola species group have the
basal tergite bands also present (medially joined in R. foliates); the anal crest of V-type
following morphological characters:
(except in R. phaselus the analsp.tergite
and R. ferringtoni bands
n.); superior developed
volsella and ofextension
with posterior V-type, and
the basal tergite bands
(except also present
in R. lamellatus, (medially
R. quadratus joined
and R. in R. median
spinicornus); foliates); the anal
volsella crestshort,
relatively of V-type
often not reaching the apex of the superior volsella (except in R. photophilus and R. remus)
and with apical plate(s); gonostylus abruptly tapered in apical portion or at the base
(emended diagnosis) and often with parallel sided apical portion [6,9].

3.2.1. Rheotanytarsus falcipedius Kyerematen, Andersen & Sæther, 2000

Diversity 2022, 14, 383 4 of 16

(except in R. phaselus and R. ferringtoni sp. n.); superior volsella with posterior extension
(except in R. lamellatus, R. quadratus and R. spinicornus); median volsella relatively short,
often not reaching the apex of the superior volsella (except in R. photophilus and R. remus)
and with apical plate(s); gonostylus abruptly tapered in apical portion or at the base
(emended diagnosis) and often with parallel sided apical portion [6,9].

3.2.1. Rheotanytarsus falcipedius Kyerematen, Andersen & Sæther, 2000

(Figures 1a and 3)
Rheotanytarsus falcipedius Kyerematen, Andersen & Sæther, 2000: 241.
Specimens examined. Two males, China, Guangdong, Ruyuan Yao Autonomous
County, Nanling National Nature Reserve, 24.903◦ N, 113.048◦ E, 688 m a.s.l., 27.VIII.2020,
light tarp, leg. X. L. Lin (BOLD sample ID and NKU: NLCH97, NLCH105); one male,
China, Guangdong, Ruyuan Yao Autonomous County, Nanling National Nature Reserve,
Laopengyidui, 24.928◦ N, 113.018◦ E, 1020 m a.s.l., 27.VIII.2020, light tarp, leg. X. L. Lin
(BOLD sample ID and NKU: NLCH124).
Diagnosis. The adult male can be distinguished from known species of Rheotanytarsus
by the following combination of characters: antenna with 13 flagellomeres; anal tergite
bands of V-type, well developed and separated; anal point constricted in the middle, with
rounded apex, and anal crests developed; superior volsella with pronounced apical and
inner extensions; digitus with a triangular projection apically, and with one seta placed on
the tubercle in the middle; median volsella of Z-type; gonostylus abruptly tapered apically
and slightly curved.
Description. Adult male (n = 3). Total length 2.12–2.31, 2.18 mm. Wing length 1.22–1.53,
1.44 mm. Total length/wing length 1.51–1.73, 1.69. Wing length/length of profemur
1.70–2.24, 1.92.
Coloration (Figure 1a). Thorax and abdomen yellow, legs brown.
Head. Antenna with 13 flagellomeres, ultimate flagellomere 253–277, 265 µm long. AR 0.54–
0.61, 0.59. Temporal setae 6–7, 7. Clypeus with 15–16, 16 setae. Tentorium
91–95, 93 µm long, 17–24, 20 µm wide. Palpomere lengths (in µm): 24–29, 27; 26–28, 27;
85–92, 89; 91–95, 93; the fifth used for DNA extraction. Third palpomere with 2 sensilla
clavata distally.
Thorax. Dorsocentrals 6–7, 7; acrostichals 7–8, 8; prealars 1. Scutellum with 6 setae.
Halteres with 5–6, 6 setae.
Wing (Figure 3a). VR 1.46–1.51, 1.48. Brachiolum with one seta, Sc bare, R with 15–18,
16 setae, R1 with 21–25, 23 setae, R4+5 with 38–44, 41 setae, M1+2 with 33–37, 35 setae, M3+4
with 21–28, 25 setae, false vein with 62–68, 65 setae, Cu with 11–12, 12 setae, Cu1 with
14–15, 15 setae, PCu with 30–35, 32 setae, An with 21–29, 24 setae, remaining veins bare.
Cell r4+5 with c. 200 setae, m with 2–3, 3 setae, m1+2 with c. 200 setae, m3+4 with c.110 setae,
cu+an with c. 100 setae.
Legs. Fore leg bearing single tibial spur. Combs of mid tibias well separated, each
bearing a spur. Tarsomere 1 of mid leg with 3–4, 4 sensilla chaetica. Lengths (in µm) and
proportions of legs as in Table 1.

Table 1. Lengths (in µm) and proportions of legs of Rheotanytarsus falcipedius Kyerematen, Andersen
& Sæther, 2000, adult male (n = 3).

fe ti ta1 ta2 ta3

P1 688–683, 674 334–347, 339 808–826, 817 407–427, 411 322–345, 330
P2 642–659, 648 432–458, 449 253–272, 267 129–141, 130 87–98, 91
P3 679 (1) - - - -
ta4 ta5 LR BV SV
P1 275–296, 283 119–131, 123 2.38–2.42, 2.41 1.55–1.61, 1.60 1.24–1.25, 1.24
P2 51–63, 57 42–52, 46 0.59 3.92–4.29, 4.21 4.11–4.25, 4.16
P3 - - - - -
Diversity 2022, 14, 383 5 of 16

Hypopygium (Figure 3b–e). Tergite IX 86–90, 88 µm long, with 2–3, 3 median setae at
the base of anal point, anal tergal bands of V-type, well developed and separated. Anal
point 27–32, 30 µm long, constricted in the middle, bearing 2–3, 3 lateral setae on each side;
crests of V-type, well developed. Transverse sternapodeme 24–33, 29 µm long, with oral
projections. Phallapodeme 60–63, 62 µm long. Gonocoxite 85–93, 90 µm long. Gonosty-
lus 72–77, 75 µm long, abruptly tapered and slightly curved apically. Superior volsella
(Figure 3d) 33–39, 35 µm long, with apical and inner extensions, with two anteromedian
setae and four dorsal setae. Digitus with triangular projections, bearing one seta located on
cylindrical tubercle in the middle. Median volsella (Figure 3e) of Z-type, 24–32, 28 µm long,
relatively short, not reaching the apex of superior volsella, with two short subulate setae
fused into a narrow plate. Inferior volsella 48–53, 50 µm long, slightly swollen apically,
with microtrichia. HR 1.18–1.21, 1.20. HV 2.91–3.00, 2.94.
Remarks. R. falcipedius is recorded in China for the first time. The Chinese specimens
fit well with the original description [7].
Diversity 2022, 14, x FOR PEER REVIEW 6 of 17
Distribution. China (Guangdong) and Thailand.

Figure
Figure 3. 3. Rheotanytarsus
Rheotanytarsus falcipedius:
falcipedius: (a)(a) wing;
wing; (b)(b) hypopygium,
hypopygium, dorsal
dorsal view;
view; (c) hypopyg- ventral
(c) hypopygium,
ium, ventral view; (d) superior volsella; (e)
view; (d) superior volsella; (e) median volsella. median volsella.

3.2.2. Rheotanytarsus ferringtoni Lin & Yao sp. n.

http://zoobank.org/NomenclaturalActs/049C6E91-FA03-4FA4-9C5A-
49BA0AF478ED (accessed on May 11, 2022).
(Figures 1b and 4)
Type material. Holotype: male, China, Yunnan, Baoshan, Longyang, Mangkuan,
Diversity 2022, 14, 383 6 of 16

3.2.2. Rheotanytarsus ferringtoni Lin & Yao sp. n.

http://zoobank.org/NomenclaturalActs/049C6E91-FA03-4FA4-9C5A-49BA0AF478ED
(accessed on 30 March 2022).
(Figures 1b and 4)
Type material. Holotype: male, China, Yunnan, Baoshan, Longyang, Mangkuan, Gaoligong-
shan National Nature Reserve, 25.3105556◦ N, 98.795000◦ E, 1475 m a.s.l., 22.V.2018, light trap,
leg. X.-L. Lin (BOLD Sample ID and NKU: XL916).
Etymology. Named after Prof. Leonard C. Ferrington Jr., for his outstanding contribu-
tion to the knowledge of Chironomidae; noun in nominative case.
Diagnosis. The adult male can be distinguished from known species of Rheotanytarsus
by the following combination of characters: antenna with 13 flagellomeres, and AR 0.43;
LR1 2.25; anal tergite bands of V-type, well developed and separated; anal point slightly
constricted in the middle, with rounded apex, and anal crests basally fused to form an arc
and apically opened; digitus with wavy inner margin, and with one seta placed on the
tubercle in the middle; superior volsella with a pronounced eagle’s beak-like extension;
median volsella of Z-type; gonostylus abruptly tapered at the base, and slightly curved.
Description. Adult male (n = 1). Total length 2.44 mm. Wing length 1.44 mm. Total
length/wing length 1.69. Wing length/length of profemur 1.90.
Coloration (Figure 1b). Thorax and legs brown, abdomen pale yellow.
Head. Antenna with 13 flagellomeres, ultimate flagellomere 232 µm long. AR 0.43.
Temporal setae 7. Clypeus with 15 setae. Tentorium 93 µm long, 20 µm wide. Palpomere
lengths (in µm): 31, 29, 113, 105, 212. Pm5/Pm3 1.88. Third palpomere with 2 sensilla
clavata distally.
Thorax. Dorsocentrals 8; acrostichals 10; prealars 1. Scutellum with 6 setae. Halteres
with 8 setae.
Wing (Figure 4a). VR 1.45. Brachiolum with one seta, Sc bare, R with 21 setae, R1 with
28 setae, R4+5 with 64 setae, RM with one seta, M1+2 with 64 setae, M3+4 with 38 setae, false
vein with 89 setae, Cu with 21 setae, Cu1 with 24 setae, PCu with 52 setae, An with 30 setae,
remaining veins bare. Cell r4+5 with c. 200 setae, m with six setae, m1+2 with c. 200 setae,
m3+4 with 130 setae, cu+an with 127 setae.
Legs. Fore leg bearing single tibial spur, 23 µm long. Combs of mid tibia 22 µm wide
with 21 µm long spur, and 17 µm wide with 31 µm long spur; combs of hind tibia 21 µm
wide with 36 µm long spur, 29 µm wide with 39 µm long spur. Tarsomere 1 of mid leg with
five sensilla chaetica. Lengths (in µm) and proportions of legs as in Table 2.

Table 2. Lengths (in µm) and proportions of legs of Rheotanytarsus ferringtoni Lin & Yao sp. n., adult
male (n = 1).

fe ti ta1 ta2 ta3 ta4 ta5 LR BV SV BR

P1 758 414 932 451 361 326 139 2.25 1.65 1.26 2.14
P2 715 524 322 169 115 72 52 0.61 3.83 3.85 4.75
P3 780 641 509 280 256 177 83 0.79 2.42 2.79 5.63

Hypopygium (Figure 4b–e). Tergite IX 78 µm long, with four median setae at the base of
anal point, anal tergal bands of V-type, well developed and separated. Anal point 38 µm long,
slightly constricted in the middle, bearing three lateral setae on each side; crests basally circular
and apically opened. Transverse sternapodeme 39 µm long, with oral projections. Phallapodeme
72 µm long. Gonocoxite 105 µm long. Gonostylus 88 µm long, gradually tapered and slightly
curved, with an oval setose area on the ventral side. Superior volsella (Figure 4d) 39 µm long,
with an apical eagle’s beak-like extension, with two anteromedian setae and four dorsal setae.
Diversity 2022, 14, 383 7 of 16

Digitus with wavy inner margin, bearing one seta located on cylindrical tubercle at the middle.
Median volsella of Z-type (Figure 4e), 33 µm long, relatively short, not reaching the apex of
inferior volsella, with three short subulate setae fused into a narrow oval plate. Inferior volsella
61 µm long, basally pronounced swollen and constricted in the middle, with microtrichia. HR
1.19. HV 2.77.
Female and immatures unknown.
Remarks. The new species resembles Rheotanytarsus falcipedius Kyerematen, Andersen
& Sæther, 2000, by having similar shapes of superior volsella and median volsella, but
can be separated from the latter species by the following combination characters: AR
0.43; superior volsella with a straight inner margin; gonostylus abruptly tapered at the
base; whereas AR 0.52–0.57, superior volsella with an inner extension; gonostylus abruptly
Diversity 2022, 14, x FOR PEER REVIEW 8 of 17
tapered in apical portion in R. falcipedius.

Figure4.
Figure 4. Rheotanytarsus
Rheotanytarsus ferringtoni sp.sp.
ferringtoni n.: n.:
(a) (a)
wing; (b) hypopygium,
wing; dorsaldorsal
(b) hypopygium, view; (c) hypopygium,
view; (c) hypopygium,
ventral view; (d) superior volsella; (e) median volsella.
ventral view; (d) superior volsella; (e) median volsella.
Diversity 2022, 14, 383 8 of 16

3.2.3. Rheotanytarsus fluminis Kawai & Sasa, 1985

(Figures 1c and 5)
Rheotanytarsus fluminis Kawai & Sasa 1985: 20.
Examined specimens. One male, China, Hainan, Lingshui, Diaoluo Mountain Forestry
Bureau, Nanxi Forest Farm, 18.66862◦ N, 109.92361◦ E, 22.IV.2008, light trap, leg. Y. Fu
(BOLD sample and NKU: DL09).
Diagnosis. The adult male can be distinguished from known species of Rheotanytarsus
by the following combination of characters: antenna with 13 flagellomeres; anal point with
parallel-sided apex; digitus rectangular, and with one seta in the middle; superior volsella
with a beak-like extension; median volsella markedly curved, with an oblong plate apically;
inferior volsella curved and apically swollen; gonostylus curved and abruptly tapered in
apical portion.
Description. Adult male (n = 1). Total length 1.54 mm. Wing length 1.09 mm. Total
length/wing length 1.41. Wing length/length of profemur 1.65.
Coloration (Figure 1c). Thorax, abdomen and legs yellowish green.
Head. Temporal setae 7. Tentorium 89 µm long, 31 µm wide. Palpomere lengths (in
µm): 28, 25, 88, 107, 193. Pm5/Pm3 2.19. Third palpomere with 2 sensilla clavata distally.
Antennas lost during collection.
Thorax. Dorsocentrals 7; acrostichals 9; prealars 1. Scutellum with 4 setae. Halteres
with 6 setae.
Wing (Figure 5a). VR 1.61. Brachiolum with one seta, Sc bare, R with 16 setae, R1 with
22 setae, R4+5 with 39 setae, RM with one seta, M1+2 with 39 setae, M3+4 with 25 setae, false
vein with 77 setae, Cu with 14 setae, Cu1 with 16 setae, PCu with 43 setae, An with 16 setae,
remaining veins bare. Cell r4+5 with c. 230 setae, m with nine setae, m1+2 with c. 250 setae,
m3+4 with c. 110 setae, cu+an with c. 130 setae.
Legs. Fore leg bearing single tibial spur, 29 µm long. Both combs of mid tibia and both
combs of hind tibia with a spur. Lengths (in µm) and proportions of legs as in Table 3.

Table 3. Lengths (in µm) and proportions of legs of Rheotanytarsus fluminis Kawai & Sasa 1985, adult
male (n = 1).

fe ti ta1 ta2 ta3 ta4 ta5 LR BV SV

P1 661 293 - - - - - - - -
P2 602 424 - - - - - - - -
P3 660 516 376 212 152 191 119 0.73 2.30 3.13

Hypopygium (Figure 5b–e). Tergite IX 86 µm long, with one median setae at the base
of anal point, anal tergal bands of V-type and separated. Anal point 36 µm long with
parallel-sided apex; crests of V-type. Transverse sternapodeme 37 µm long, with oral
projections. Phallapodeme 34 µm long. Gonocoxite 99 µm long. Gonostylus 88 µm long,
abruptly tapered distally and curved. Superior volsella (Figure 5d) 30 µm long, with an
apical beak-like extension, with two anteromedian setae and four dorsal setae. Digitus
broad and rectangular, bearing one seta located in the middle. Median volsella (Figure 5e)
48 µm long, markedly curved and not reaching the apex of inferior volsella, with subulate
setae and four subulate setae fused into an oblong plate. Inferior volsella 62 µm long,
curved and swollen apically, with microtrichia. HR 1.13. HV 1.74.
Remarks. R. fluminis is recorded in China for the first time. The Chinese specimen fits
well with the original description [30].
Distribution. Japan and China (Hainan).
Diversity 2022, 14, 383 9 of 16
Diversity 2022, 14, x FOR PEER REVIEW 10 of 17

Figure5.
Figure 5. Rheotanytarsus
Rheotanytarsus fluminis: (a)(a)
fluminis: wing; (b) (b)
wing; hypopygium, dorsal
hypopygium, view;view;
dorsal (c) hypopygium, ventral ventral
(c) hypopygium,
view;(d)
view; (d) superior
superior volsella;
volsella;(e)
(e)median
median volsella.
volsella.

3.2.4. Rheotanytarsus
3.2.4. Rheotanytarsus illiesi
illiesiSiebert, 1979
Siebert, 1979
(Figures 1d
(Figures 1d and
and6)6)
Rheotanytarsusilliesi
Rheotanytarsus illiesiSiebert, 1979:
Siebert, 1979:165.
165.
Examined specimens. One male, China, Inner Mongolia, Hulumbuir, Honghuaer’ji,
Examined specimens. One male, China, Inner Mongolia, Hulumbuir, Honghuaer’ji,
49.930766° N, 123.567751° E, 428 m a.s.l. 2.Ⅷ.2016, light trap, leg. C. Song (BOLD sample
49.930766◦ N, 123.567751◦ E, 428 m a.s.l. 2.VIII.2016, light trap, leg. C. Song (BOLD sample
ID and NKU: XL1675).
ID and NKU: XL1675).
Diagnosis. The adult male can be distinguished from known species of Rheotanytarsus
Diagnosis.
by the followingThe adult male
combination can be distinguished
of characters: antenna with from known species
13 flagellomeres andof Rheotanytarsus
with rela-
by the following combination of characters: antenna with 13 flagellomeres
tively high antennal ratio; inner tibial comb of hind tibia with a spur; anal point slightly and with
relatively
swollen apically; superior volsella with a relatively blunt projection; median volsellaslightly
high antennal ratio; inner tibial comb of hind tibia with a spur; anal point
swollen
markedlyapically; superior
curved, with volsella
an elongate platewith a relatively
apically; blunt projection;
inferior volsella median
curved; gonostylus ab-volsella
markedly curved, with an
ruptly tapered in apical portion. elongate plate apically; inferior volsella curved; gonostylus
abruptly tapered Adult
Description. in apical
male portion.
(n = 1). Total length 3.11 mm. Wing length 1.76 mm. Total
length/wing
Description. Adult malelength/length
length 1.77. Wing (n = 1). Total oflength
profemur
3.111.85.
mm. Wing length 1.76 mm. Total
length/wing length 1.77. Wing length/length of profemur 1.85.
Coloration (Figure 1d). Thorax and legs brown, abdomen, pale yellow.
Head. Antenna with 13 flagellomeres, ultimate flagellomere 534 µm long. AR 0.99.
Temporal setae 10. Clypeus with 17 setae. Tentorium 124 µm long, 26 µm wide. Palpomere
Diversity 2022, 14, 383 10 of 16

lengths (in µm): 42, 43, 132, 134; the fifth used for DNA extraction. Third palpomere with
2 sensilla clavata distally.
Thorax. Dorsocentrals 9; acrostichals 9; prealars 1. Scutellum with 10 setae. Halteres
with 7 setae.
Wing (Figure 6a). VR 1.34. Brachiolum with one seta, Sc bare, R with 26 setae, R1 with
32 setae, R4+5 with 61 setae, M1+2 with 35 setae, M3+4 with 35 setae, false vein with 93 setae,
Cu with 22 setae, Cu1 with 28 setae, PCu with 61 setae, An with 32 setae, remaining veins
bare. Cell r4+5 with c. 300 setae, m with 20 setae, m1+2 with c. 250 setae, m3+4 with c.
200 setae, cu + an with c. 150 setae.
Legs. Fore leg bearing single tibial spur. Combs of mid and hind tibias well separated,
each
Diversity 2022, 14, x FOR PEER REVIEW
bearing a spur. Tarsomere 1 of mid leg with four sensilla chaetica. Lengths
12 of 17
(in µm)
and proportions of legs as in Table 4.

Figure6.6.Rheotanytarsus
Figure Rheotanytarsus illiesi:
illiesi: (a)
(a) wing;
wing; (b)
(b) hypopygium,
hypopygium,dorsal
dorsalview;
view;(c)
(c)hypopygium,
hypopygium,ventral
ventral view;
view; (d) superior volsella.
(d) superior volsella.
3.2.5. Rheotanytarsus muscicola Thienemann, 1929
(Figure 1e)
Rheotanytarsus muscicola Thienemann, 1929: 114; Lehmann 1970: 362; Reiss 1971: 208,
Wang & Zheng 1993: 90; Wang & Guo 2004: 9.
Specimens examined. One male, China, Liaoning, Shenyang, Benxi, Wangtian Cave
Scenic Area, 41.192° N, 125.267° E, 254 m a.s.l., 2.Ⅸ.2014, light tarp, leg. C Song (BOLD
sample ID and NKU: XL228). One male, China, Hebei, Baoding, Baoding, Zijingguan,
Juma River, 39.428° N, 115.17° E, 521 m a.s.l., 8.Ⅴ.2018, light tarp, leg. X. L. Lin (BOLD
sample ID and NKU: XL1344).
Diversity 2022, 14, 383 11 of 16

Table 4. Lengths (in µm) and proportions of legs of Rheotanytarsus illiesi Siebert 1979, adult male (n = 1).

fe ti ta1 ta2 ta3 ta4 ta5 LR BV SV

P1 954 507 1189 547 425 343 138 2.35 1.82 1.23
P2 923 688 405 205 127 96 66 0.59 4.08 3.98
P3 1016 867 629 369 292 189 107 0.73 2.62 2.99

Hypopygium (Figure 6b–d). Tergite IX 113 µm long, with eight around the base of anal
point and two setae covered by the anal point, anal tergal bands of V-type and separated.
Anal point 43 µm long constricted in the middle; crests with two separated arcs. Transverse
sternapodeme 38 µm long, with pronounced oral projections. Phallapodeme 90 µm long.
Gonocoxite 121 µm long. Gonostylus 127 µm long, abruptly tapered distally. Superior
volsella (Figure 6d) 44 µm long, with a blunt projection, with two anteromedian setae and
four (left) or six (right) dorsal setae. Digitus thumb-shaped, bearing one seta located on
cylindrical tubercle in the middle. Median volsella 59 µm long, markedly curved, nearly
reaching the apex of superior volsella, with an oblong plate densely covered setae on the
ventral side. The plate was well illustrated in the original descriptive [10]. Inferior volsella
77 µm long, curved with microtrichia and ten setae apically. HR 0.95. HV 2.45.
Remarks. The adult male of R. illiesi is recorded in China for the first time. The Chinese
specimen fits well with the original description [10], but the body size is smaller than the
German specimens. A larva from Xinjiang was associated with the adult male (Figure 2) by
DNA barcode, but was misidentified as R. muscicola morphologically and without access to
examine this voucher.
Distribution. Germany and China (Inner Mongolia, Xinjiang).

3.2.5. Rheotanytarsus muscicola Thienemann, 1929

(Figure 1e)
Rheotanytarsus muscicola Thienemann, 1929: 114; Lehmann 1970: 362; Reiss 1971: 208,
Wang & Zheng 1993: 90; Wang & Guo 2004: 9.
Specimens examined. One male, China, Liaoning, Shenyang, Benxi, Wangtian Cave
Scenic Area, 41.192◦ N, 125.267◦ E, 254 m a.s.l., 2.IX.2014, light tarp, leg. C Song (BOLD
sample ID and NKU: XL228). One male, China, Hebei, Baoding, Baoding, Zijingguan, Juma
River, 39.428◦ N, 115.17◦ E, 521 m a.s.l., 8.V.2018, light tarp, leg. X. L. Lin (BOLD sample ID
and NKU: XL1344).
Diagnosis. This species can be separated from other members of the group by the
following combination of characters: inner tibial comb of hind tibia without a spur; median
volsella of S-type; and the base of anal point with only one seta.
Distribution. Europe, north Africa, Canada, China (Fujian, Guangdong, Hebei, Liaon-
ing, Shaanxi, Shandong, Yunnan).

3.2.6. Rheotanytarsus Photophilus Goetghebuer, 1921

(Figure 1f)
Rheotanytarsus photophilus Goetghebuer, 1921: 115; Lehmann 1970: 365.
Specimens examined. One male, China, Jilin, Dunhua, Liuding mountain scenic area,
16. VII.2016, light trap, leg. C. Song (BOLD sample & NKU: XL3678).
Diagnosis. The adult male can be distinguished from known species of Rheotanytarsus
by the following combination of characters: antenna with 13 flagellomeres and with
relatively high antennal ratio; inner tibial comb of hind tibia with a spur; superior volsella
with a projection; median volsella markedly of S-type, nearly reaching the apex of inferior
volsella; inferior volsella slightly curved; gonostylus abruptly tapered in apical portion.
Description. Adult male (n = 1). Total length 2.95 mm. Wing length 1.73 mm. Total
length/wing length 1.71 Wing length/length of profemur 1.90.
Coloration (Figure 1d). Thorax yellow with brown stripes, abdomen and legs pale yellow.
Diversity 2022, 14, 383 12 of 16

Head. Antenna with 13 flagellomeres, ultimate flagellomere 525 µm long. AR 1.03.

Temporal setae 10. Clypeus with 20 setae. Tentorium 130 µm long, 33 µm wide. Palpomere
lengths (in µm): 35, 40, 125, 125, 245. Third palpomere with 2 sensilla clavata distally.
Thorax. Dorsocentrals 10; acrostichals 11; prealars 1. Scutellum with 4 setae. Halteres
with 2 setae.
Legs. Fore leg bearing single tibial spur. Combs of mid and hind tibias well separated,
each bearing a spur. Tarsomere 1 of mid leg with three sensilla chaetica. Lengths (in µm)
and proportions of legs as in Table 5.

Table 5. Lengths (in µm) and proportions of legs of Rheotanytarsus photophilus Goetghebuer, 1921,
adult male (n = 1).

fe ti ta1 ta2 ta3 ta4 ta5 LR BV SV

P1 910 480 1035 510 410 310 130 2.16 1.78 1.34
P2 900 680 380 200 160 100 60 0.56 3.77 4.16
P3 1010 850 - - - - - - - -

Hypopygium. Anal point 40 µm long constricted in the middle. Transverse ster-

napodeme 48 µm long. Phallapodeme 85 µm long. Gonocoxite 125 µm long. Gonostylus
115 µm long, abruptly tapered distally. Superior volsella with a projection, with two an-
teromedian setae and four dorsal setae. Median volsella 70 µm long, relatively long and
of S-type, nearly reaching the apex of inferior volsella, with distinct flaky setae apically.
Inferior volsella 75 µm long, slightly curved and with microtrichia. HR 1.09. HV 2.57.
Remarks. R. photophilus is recorded in China for the first time. The Chinese specimen
fits well with the description of the revision by Lehmann [3]. The hypopygium has been
well illustrated by Lehmann [3]. R. photophilus shows a large intraspecific divergence (up to
7%) in COI barcode (Figure 2).
Distribution. Belgium, Finland, Germany and China (Jilin).

3.2.7. Rheotanytarsus quadratus Wang & Guo, 2004

Rheotanytarsus quadratus Wang & Guo, 2004: 9; Hazra et al. 2016: 12.
Specimens examined. Type materials: Holotype, male, China, Fujian, Nanping, Maodi,
Mangdangshan, 23.IX.2002, light tarp, leg. Z. Liu (NKU: 20805). Paratype, male, China,
Fujian, Yongtai, 17.IX.2002, light tarp, leg. Z. Liu (NKU: 20395).
Diagnosis. This species can be separated from other members of the group by the
following combination of characters: relatively high AR; superior volsella rectangular.
Distribution. China (Fujian).
Diversity 2022, 14, 383 13 of 16

3.3 Key to Known Adult Males of the Rheotanytarsus Muscicola Species Group
1 Superior volsella without a posterior extension 2
_ Superior volsella with a posterior extension 4
2 Superior volsella rectangular R. quadratus Wang & Guo
_ Superior volsella oval or ovoid 3
3 AR 0.31–0.38; median volsella with subulate setae fused into a plate R. spinicornus Hazra, Brahma & Sanyal
_ AR 0.45–0.87; median volsella with lamelliform setae fused into a plate R. lamellatus Reiss
4 Median volsella relatively long, reaching the apex of inferior volsella 5
_ Median volsella relatively short, not reaching the apex of inferior volsella 6
5 Digitus rectangular and reaching beyond the superior volsella; median volsella slightly curved R. remus Kyerematen a& Sæther
_ Digitus triangular and not reaching beyond the superior volsella; median volsella of S-type R. photophilus Goetghebuer
6 Superior volsella reniform with digitiform extension R. phaselus Kyerematen, Andersen & Sæther
_ Superior volsella not reniform 7
7 Median volsella slightly or not curved 8
_ Median volsella markedly curved 9
8 Superior volsella subquadrangular; anal point with parallel-sided apex R. kusii Kyerematen
_ Superior volsella subtriangular; anal point constricted in the middle R. foliatus Kyerematen & Andersen
9 Median volsella of L-type R. guanacastensis Kyerematen & Andersen
_ Median volsella of other types 10
10 Apical plate of median volsella absent R. thunesi Kyerematen & Andersen
_ Apical plate(s) of median volsella present 11
11 Apical portion of gonostylus relatively thin 12
_ Apical portion of gonostylus relatively thick 14
12 Anal point with parallel-sided apex, gonostylus curved apically R. fluminis Kawai & Sasa
_ Anal point constricted in the middle; gonostylus not curved apically 13
13 AR 0.86–1.04; median volsella with simple setae fused into an egg-shaped plate R. illiesi Siebert
_ AR 0.36–0.39; median volsella with lamelliform setae fused into a broad plate R. subtilis Kyerematen & Andersen
14 Gonostylus abruptly tapered at the base R. ferringtoni Lin & Yao sp. n.
_ Gonostylus abruptly tapered in apical portion 15
15 The extension of superior volsella relatively blunt; the inner tibial comb of the hind leg without a spur R. muscicola Thienemann
_ The extension of superior volsella relatively sharp; the inner tibial comb of the hind leg with a spur R. falcipedius Kyerematen, Andersen & Sæther
Diversity 2022, 14, 383 14 of 16

4. Conclusions
Our study shows concordance between morphological species concepts and DNA
barcodes. After reviewing the Rheotanytarsus muscicola species group from China using
DNA barcodes, we detected and described one new species and four species new to China,
and provided updated keys to the adult male.

Author Contributions: Methodology, X.-L.L.; software, Y.Y. and W.-B.L.; investigation, X.-L.L. and
Y.Y.; data curation, X.-L.L.; writing—original draft preparation, X.Q. and Y.Y.; writing—review and
editing, C.-C.Y., X.-L.L. and X.-H.W.; supervision, C.-C.Y., X.-L.L. and X.-H.W.; project administration,
X.-L.L.; funding acquisition, X.Q. and X.-L.L. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China, grant
numbers 32070481, 32170473, and 31900344, and the Zhejiang Provincial Natural Science Foundation
of China, grant number LY22C040003.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: A list of all species, specimens, their individual images, georeferences,
primers, sequences and other relevant laboratory data of all 22 specimens are available through the
dataset “DNA bar-codes of Rheotanytarsus muscicola species group (DS-MUSCICO)” in the Barcode of
Life Data System (http://www.boldsystems.org, BOLD (accessed on 30 March 2022)).
Acknowledgments: We are grateful to the Canadian Centre for DNA Barcoding (CCDB, Guelph,
Ontario, Canada) for DNA sequencing.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

An anal vein
an cell below anal vein
AR antennal ration, ratio of length of apical elongated flagellomere plus any flagell-
omeres distal to it divided by combined length of the more basal flagellomeres
BV Bein-Verhältnis ratio, length of (femur + tibia + ta1 )/length of (ta2 + ta3 + ta4 + ta5 )
Cu cubitus
cu cell below Cu vein
Cu1 cell below Cu1 vein
fe femur
HR hypopygium ration, length of gonocoxite/length of gonostylus
HV hypopygium value, total length/length of gonostylus times ten
LR leg ration, length of tarsomere 1/length of tibia
m cell below median vein
M, M1+2 , M3+4 media and its branches
m1+2 cell below M1+2 vein
m3+4 cell below M3+4 vein
P1 fore leg
P2 mid leg
P3 hind leg
PCu postcubital vein
R Radius
R1 Radius 1 vein
R4+5 Radius 4 + 5 vein
RM apparent radius to media cross vein
r4+5 cell below R4+5 vein
Sc subcosta
SV Schenkel-Schiene-Verhältnis ratio, length of (femur + tibia)/length of ta1
ti tibia
Diversity 2022, 14, 383 15 of 16

ta1 tarsomere 1
ta2 tarsomere 2
ta3 tarsomere 3
ta4 tarsomere 4
ta5 tarsomere 5
VR Venarum ration, length of Cubitus/length of Media

References
1. Wang, X.-H.; Zheng, L.-Y. Taxonomic studies on Chironomidae from China VI. Genus Rheotanytarsus Thienemann & Bause
(Diptera: Chironomidae). Acta Sci. Nat. Univ. Nankaiensis 1993, 1, 89–93.
2. Wang, X.-H.; Guo, Y.-H. A review of the genus Rheotanytarsus Thienemann & Bause from China (Diptera: Chironomidae:
Tanytarsini). Zootaxa 2004, 650, 1–19.
3. Lehmann, J. Revision der europäischen Arten (Imagines♂♂und Puppen♂♂) der Gattung Rheotanytarsus BAUSE (Diptera,
Chironomidae). Zool Anz 1970, 185, 343–378.
4. Moubayed, Z. Chironomids from running waters of Thailand: Description of Rheotanytarsus thailandensis sp.n. and Tanytarsus
thaicus sp.n. (Dipt., Chironomidae). Hydrobiologia 1990, 203, 29–33. [CrossRef]
5. Yao, Y.; Liu, W.-B.; Wang, X.-H.; Lin, X.-L. Rheotanytarsus baihualingensis and R. diaoluoensis (Diptera: Chironomidae), two new
species from Oriental China. Ann. Zool. Fenn. 2022, 59, 149–154. [CrossRef]
6. Hazra, N.; Brahma, S.; Sanyal, K. New Species of Rheotanytarsus Thienemann and Bause (Diptera: Chironomidae: Tanytarsini)
from Darjeeling–Sikkim, Himalaya, India, with Revised Keys to the Adult Males and Pupae of the Species of the Oriental Region.
Psyche 2016, 2016, 1–14. [CrossRef]
7. Kyerematen, R.A.; Sæther, O.A. A review of Afrotropical Rheotanytarsus Thienemann et Bause, 1913 (Diptera: Chironomidae).
Tijdschr. Voor Entomol. 2000, 143, 27–70. [CrossRef]
8. Cranston, P.S. Revision of Australian Rheotanytarsus Thienemann & Bause (Diptera: Chironomidae), with emphasis on immature
stages. Invertebr. Syst. 1997, 11, 705–734.
9. Sæther, O.A.; Kyerematen, R.A. Towards phylogeny and zoogography of the genus Rheotanytarsus Thienemann et Bause, 1913
(Diptera: Chironomidae). Tijdschr. Voor Entomol. 2001, 144, 73–117. [CrossRef]
10. Siebert, M. Two new chironomids (Diptera: Chironomidae) from Germany and Austria. Aquat. Insects 1979, 1, 165–168. [CrossRef]
11. Reiss, F. Ein Beitrag zur ostpalaearktischen Chironomidenfauna (Diptera) am Beispiel einiger Tanytarsini-Arten aus der Mongolei
und Ostsibirien. Entomol. Tidskr. 1971, 92, 198–212.
12. Moubayed-Breil, J.; Langton, P.H.; Ashe, P. Rheotanytarsus dactylophoreus, a new mountain species from streams in the Eastern
Pyrenees and Corsica (Diptera: Chironomidae). Fauna Nor. 2012, 31, 167. [CrossRef]
13. Epler, J.H.; Ekrem, T.; Cranston, P.S. The larvae of Chironominae (Diptera: Chironomidae) of the Holarctic region—keys and
diagnoses. In Chironomidae of the Holarctic Region: Keys and Diagnoses, Part 1: Larvae; Cederholm, L., Ed.; Insect Systematics and
Evolution, Supplement: Lund, Sweden, 2013; Volume 66, pp. 387–556.
14. Kyerematen, R.A.K.; Andersen, T. Rheotanytarsus Thienemann et Bause (Diptera: Chironomidae) from Central America and
Mexico. Stud. Neotrop. Fauna Environ. 2002, 37, 23–51. [CrossRef]
15. Hebert, P.D.N.; Cywinska, A.; Ball, S.L. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B Biol. Sci. 2003, 270,
313–321. [CrossRef]
16. Hebert, P.D.N.; Ratnasingham, S.; deWaard, J.R. Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among
closely related species. Proc. R. Soc. Lond. B Biol. Sci. 2003, 270, S96–S99. [CrossRef]
17. Lin, X.-L.; Stur, E.; Ekrem, T. Exploring genetic divergence in a species-rich insect genus using 2790 DNA Barcodes. PLoS ONE
2015, 10, e0138993. [CrossRef]
18. Lin, X.-L.; Stur, E.; Ekrem, T. DNA barcodes and morphology reveal unrecognized species of Chironomidae (Diptera). Insect Syst.
Evol. 2018, 49, 329–398. [CrossRef]
19. Lin, X.-L.; Yu, H.-J.; Wang, X.-H.; Bu, W.-J.; Wang, X.-H.; Yan, C.-C.; Liu, W.-B. New or little-known Boreoheptagyia (Diptera:
Chironomidae) in China inferred from morpholgy and DNA barcodes. Zookeys 2021, 1040, 187–200. [CrossRef]
20. Lin, X.L.; Mo, L.; Bu, W.J.; Wang, X.H. The first comprehensive DNA barcode reference library of Chinese Tanytarsus (Diptera:
Chironomidae) for environmental DNA metabarcoding. Divers. Distrib. 2021, 27, 1932–1941. [CrossRef]
21. Lin, X.L.; Jiang, K.; Liu, W.B.; Liu, W.; Bu, W.J.; Wang, X.H.; Mo, L. Toward a global DNA barcode reference library of the
intolerant nonbiting midge genus Rheocricotopus Brundin, 1956. Ecol. Evol. 2021, 11, 12161–12172. [CrossRef]
22. Song, C.; Lin, X.-L.; Wang, Q.; Wang, X.-H. DNA barcodes successfully delimit morphospecies in a superdiverse insect genus.
Zool. Scr. 2018, 47, 311–324. [CrossRef]
23. Anderson, A.M.; Stur, E.; Ekrem, T. Molecular and morphological methods reveal cryptic diversity and three new species of
Nearctic Micropsectra (Diptera: Chironomidae). Freshw. Sci. 2013, 32, 892–921. [CrossRef]
24. Lin, X.-L.; Yu, H.-J.; Zhang, R.-L.; Wang, X.-H. Polypedilum (Cerobregma) heberti sp. n. (Diptera: Chironomidae) from Gaoligong
Mountains, Yunnan, China. Zootaxa 2019, 4571, 255–262. [CrossRef]
25. Lin, X.-L.; Yu, H.-J.; Wang, Q.; Bu, W.-J.; Wang, X.-H. DNA barcodes and morphology confirm a new species of Rheocricotopus
(Psilocricotopus) orientalis group (Diptera: Chironomidae). Zootaxa 2020, 4768, 282–290. [CrossRef]
Diversity 2022, 14, 383 16 of 16

26. Qi, X.; Wang, X.-H.; Andersen, T.; Lin, X.-L. A new species of Manoa Fittkau (Diptera: Chironomidae), with DNA barcodes from
Xianju National Park, Oriental China. Zootaxa 2017, 4231, 398–408. [CrossRef]
27. Gadawski, P.; Montagna, M.; Rossaro, B.; Giłka, W.; Pešić, V.; Grabowski, M.; Magoga, G. DNA barcoding of Chironomidae from
the Lake Skadar region: Reference library and a comparative analysis of the European fauna. Divers. Distrib. 2022. [CrossRef]
28. Michailova, P.; Lencioni, V.; Nenov, M.; Nikolov, S. Can DNA barcoding be used to identify closely related Clunio Haliday, 1855
species (Diptera: Chironomidae, Orthocladiinae). Zootaxa 2021, 4927, 1–8. [CrossRef] [PubMed]
29. Sæther, O.A. Some Nearctic Podonominae, Diamesinae, and Orthocladiinae (Diptera: Chironomidae). Bull. Fish. Res. Bd Can.
1969, 170, 1–154.
30. Kawai, K.; Sasa, M. Seven new species of chironomid midges (Diptera, Chironomidae) from the Ohta River, Japan. JPN J. Limnol.
(Rikusuigaku Zasshi) 1985, 46, 15–24. [CrossRef]
31. Sæther, O.A. Glossary of chironomid morphology terminology (Diptera: Chironomidae). Entomol. Scand. Suppl. 1980, 14, 1–51.
32. Folmer, O.; Black, M.; Hoeh, W.; Lutz, R.; Vrijenhoek, R. DNA primers for amplification of mitochondrial cytochrome c oxidase
subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 1994, 3, 294–299.
33. deWaard, J.R.; Ivanova, N.V.; Hajibabaei, M.; Hebert, P.D.N. Assembling DNA barcodes: Analytical protocols. In Methods in
Molecular Biology: Environmental Genetics; Cristofre, M., Ed.; Humana Press Inc.: Ottawa, ON, Canada, 2008; pp. 275–293.
34. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32,
1792–1797. [CrossRef]
35. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38,
3022–3027. [CrossRef]
36. Ratnasingham, S.; Hebert, P.D.N. BOLD: The Barcode of Life Data System (www.barcodinglife.org). Mol. Ecol. Notes 2007, 7,
355–364. [CrossRef]
37. Ratnasingham, S.; Hebert, P.D.N. A DNA-based registry for all animal species: The barcode index number (BIN) system. PLoS
ONE 2013, 8, e66213. [CrossRef]
38. Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4,
406–425.
39. Kimura, M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide
sequences. J. Mol. Evol. 1980, 16, 111–120. [CrossRef]