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https://doi.org/10.11646/zootaxa.5319.1.3
http://zoobank.org/urn:lsid:zoobank.org:pub:9F99055B-D6E7-4D7B-B2CE-81E1FD21A9AE
48 Accepted by B. Rossaro: 11 Jun. 2023; published: 24 Jul. 2023
Article ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Zootaxa 5319 (1): 048–056
https://www.mapress.com/zt/
Copyright © 2023 Magnolia Press
DNA Barcode of symbiotic chironomids: Findings in the genus Symbiocladius
(Diptera: Chironomidae)
KAREN VELÁSQUEZ-RODRÍGUEZ1,6,7, XIAO-LONG LIN2,3, PAMELA SÁNCHEZ-VENDIZÚ4,5, RAÚL
LOAYZA-MURO6, ANA HUAMANTINCO7 & NARCÍS PRAT8
1Facultad de Ciencias de la Vida y de la Salud, Universidad Científica del Sur, Lima, Perú.
�
kvelasquezr@cientifica.edu.pe; https://orcid.org/0000-0001-9540-796X
2Engineering Research Center of Environmental DNA and Ecological Water Health Assessment, Shanghai Ocean University, Shanghai
201306, China.
�
lin880224@gmail.com; https://orcid.org/0000-0001-6544-6204
3Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, Shanghai 201306,
China
4Programa de Doctorado en Ciencias mención Ecología y Evolución, Escuela Graduados, Facultad de Ciencias, Universidad Austral
de Chile, Valdivia, Chile.
�
p.sanchez.vendizu@gmail.com; https://orcid.org/0000-0002-3374-6031
5Departamento de Mastozoología, Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru
6Universidad Peruana Cayetano Heredia, Facultad de Ciencias e Ingeniería, Laboratorio de Ecotoxicología, San Martín de Porres
15102, Peru.
�
raul.loayza@upch.pe; https://orcid.org/0000-0002-1312-311X
7Laboratorio de Invertebrados Acuáticos, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
�
ahuamantincoa1@unmsm.edu.pe; https://orcid.org/0000-0001-6558-1326
8Grupo de Investigación FEHM (Freshwater Ecology Hydrology and Management), Departamento de Biología, Evolutiva, Ecología y
Ciencias Ambientales, Facultad de Biología, Universidad de Barcelona, Barcelona, Spain.
�
nprat@ub.edu; https://orcid.org/0000-0002-1550-1305
ABSTRACT
Chironomidae of symbiotic habits have been recorded in different parts of the world, among commensals and parasites.
There are different genera reported at the moment, however questions such as the origin of commensal or parasitic life,
which occurred first or what are their benefits remain debatable. In order to contribute with information to elucidate the
above mentioned issues, the present study reports the finding of immature stages of Symbiocladius (Acletus) wygodzinskyi
Roback, 1965 in the Churup stream located in the Andes Cordillera (Peru), living on nymphs of Leptophlebiidae
(Ephemeroptera). We present a morphological description of immature stages of this species and for the first time the
sequence of COX1 gene S. (A.) wygodzinskyi. The genetic result also supports differences between the morphospecies of
Symbiocladius (Symbiocladius) rhithrogenae Zavřel, 1924 and S. (A.) wygodzinskyi in 23%.
Key words: Commensalism, parasitism, COX1, taxonomy, Andes
INTRODUCTION
Commensalism is a type of symbiosis where the commensal benefits without detriment to the host. In parasitism,
the parasite benefits at the expense of the host. Chironomidae have all three modes of life (free-living, parasitic
and commensal); among the genera with symbiotic behaviors are Symbiocladius, Epoicocladius, Rheotanytarsus,
Nanocladius and Eukiefferiella. For example, Epoicocladius lives on Ephemeroptera (Svensson, 1979; Tokeshi,
1986), and a species of the subgenus Nanocladius (Plecopteracoluthus) was reported to live on Plecoptera (Dorvillé
et al., 2000; Henriques-Oliveira & Nessimian, 2009; Inoue et al., 2015), Ephemeroptera (Henriques-Oliveira &
Nessimian, 2009; Pepinelli et al., 2009) and Megaloptera (Hayashi, 1998).
FINDINGS IN THE GENUS SYMBIOCLADIUS Zootaxa 5319 (1) © 2023 Magnolia Press · 49
Epoicocladius and Symbiocladius larvae are restricted to live phoretically, with no free-living members on
any substrate (Roback, 1965; Codreanu, 1939). In contrast, most of the Nanocladius larvae are free-living, never
phoretic, only subgenus Nanocladius (Plecopteracoluthus) being symbiotic (Sæther, 1977).
The origin of symbiotic relationships (commensal and parasitic) in Chironomidae does not seem to be unique
but may have occurred several times independently in the phylogeny of the group (Steffan, 1968). This is reinforced
by reports of commensal and parasitic relationships between different genera of Chironomidae and hosts of other
groups (gastropods, bivalves and fish) (Tokeshi, 1993). However, information on symbiotic chironomids and their
relationship with their free-living relatives is scarce. As mentioned by Tokeshi (1993), the diversity of life forms
within Chironomidae provides the opportunity for comparative studies of populations, as well as on the evolutionary
course of commensal and parasitic life.
Symbiocladius Kieffer, 1925 is one of the chironomid genera that lives phoretic and parasitic on Ephemeroptera
(Hynes, 1976; Jacobsen, 1995; Prat et al., 2013). It has a wide distribution that includes North and South America,
Europe, Asia, Oceania and Australia (Tokeshi, 1993). Roback (1965) proposed the erection of two subgenera within
the genus Symbiocladius based on morphological characters of the adult and immature stages: Symbiocladius
(Acletius) Roback, 1965 and Symbiocladius (Symbiocladius) Kieffer, 1925. Species of the subgenus Acletius are
reported for the Neotropical and Australian region, while species of the subgenus Symbiocladius are reported for
the Holarctic region. At present, there are three species described within the subgenus Acletius: Symbiocladius
(Acletius) renatae Gonser & Spies, 1997, S. (A.) wygodzinskyi Roback, 1965, S. (A.) aurifodinae Hynes, 1976;
and four within the subgenus Symbiocladius: Symbiocladius (Symbiocladius) chattahoocheensis Caldwell, 1984, S.
(S.) equitans Claassen, 1922, S. (S.) rhithrogenae Zavřel, 1924 and S. (S.) villosus Makarchenko, 2015. Moreover,
the only genetic information (COX1 gene) for this genus published in the Barcode of Life Data System (BOLD)
(Ratnasingham & Hebert, 2007) corresponds to S. (S.) rhithogenae from China.
To date, the only species recorded for Peru is Symbiocladius (Acletius) wygodzinskyi (Prat et al., 2013),
which current distribution extends to southern Chile and Argentina (Gonser & Spies, 1997) but it still has scarce
information. Therefore, the goal of this study is to present a new record on S. (A.) wygodzinskyi in Peru based on
morphological and genetic data.
MATERIAL AND METHODS
Sampling of chironomid species
We collected two early instar larvae and two larvae in process of pupation of Symbiocladius (A.) wygodzinskyi
using Surber nets in Churup stream, province of Huaraz, Ancash Region, Peru (9°29’14.70” S; 77°25’58.92” W).
The Churup stream, which is a third-order stream, receives water from the Churup lagoon. Our sampling point was
located at a pool situated beneath a small waterfall, at an elevation of 4180 meters above sea level (a.s.l.) (Fig. 1A).
The environmental conditions at this location include a pH of 7.8, conductivity of 44 µS/cm, oxygen saturation of
96%, 10% particulate organic matter, and a turbidity of 0.9 NTU.
From China, adult specimens of the genus Symbiocladius were collected from different locations using sweep
nets during the afternoon hours. The collection locations in China include the Jiangkou sector of the Guizhou province
(27°42’11.16” N; 108°50’6.00” E) at an elevation of 367 meters a.s.l., Xuanen (29°40’6.96” N; 109°36’32.40” E)
at an elevation of 810 meters a.s.l., and Hefeng (29°53’33.70” N; 110°01’44.40” E) in the Hubei province at an
elevation of 1200 meters a.s.l. All collected specimens were preserved in 85% ethanol and stored at 4°C (Fig. 1).
Morphological analysis
The cephalic capsule was removed from the body prior to DNA extraction for preparation into permanent slides.
They were digested in 10% KOH at room temperature (22°C) for 12 h, then passed through a battery of alcohols
and finally mounted in Euparal medium and left to dry at room temperature for two weeks. The methodology used
followed the criteria and the mounting protocol of Epler (2001). The number of specimens or structures measured in
different species is mentioned in each case as “n”. In the case of supernumerary structures, “n” refers to that structure
VELÁSQUEZ-RODRÍGUEZ ET AL.
50 · Zootaxa 5319 (1) © 2023 Magnolia Press
and not to the specimens, unless otherwise noted. For taxonomic identification, the key of Gonser & Spies (1997)
and the description of Roback (1965) and Prat et al. (2013) were used. Digital photographs of slide specimens were
taken using a Nikon SMZ 745T stereoscopic microscope, at the Laboratory of Aquatic Invertebrates, Universidad
Nacional Mayor de San Marcos, Lima, Peru.
FIGURE 1. Records in provinces or states of Symbiocladius in the world. Records of S. wygodzinskyi and Symbiocladius
species with available COX1 sequences.
DNA extraction and amplification
DNA extraction was performed using the ZymoResearch Quick-DNA Miniprep Plus kit, with the entire body used
except for the cephalic capsule. The manufacturing instructions were followed, with a modification based on Krosch
& Cranston (2012). Initially, the bodies were digested in Proteinase K at 55°C for 3 hours. This resulted in tissue
degradation and separation of the exuviae, which was carefully removed by using fine sterile tweezers, followed by
preservation in 96% alcohol. After removing the exuviae, Proteinase K digestion continued overnight. The DNA
extraction process was then completed by following the kit manufacturer’s instructions.
A 658 bp fragment of the COX1 region was PCR-amplified using the universal primers LCO1490 and
HCO2198 (Folmer et al. 1994). DNA amplification was carried out in 25 μL total reactions using 12.5 μL KAPA
Taq ReadyMix (contains KAPA Taq DNA Polymerase, 1 U per 50 μL reaction), KAPA Taq Buffer, dNTPs (0.2 mM
of each dNTP at 1X), MgCl2 (1.5 mM at 1X) and stabilizers), 8 μL template DNA, 0.6 μL of each primer (10 μM)
and 3.3 μL of endonuclease-free H2O.
Amplification cycles were performed on an Eppendorf Mastercycler Nexus following a program with an initial
FINDINGS IN THE GENUS SYMBIOCLADIUS Zootaxa 5319 (1) © 2023 Magnolia Press · 51
denaturation step of 95°C for 3 min, followed by 39 cycles of denaturation at 95°C for 30 s, annealing at 48°C for
30 s, and extension at 72°C for 1 min, and 1 cycle of the final extension at 72°C for 1 min. PCR products were sent
to sequence at BGI (China) and Macrogen (Korea). Sequences of Symbiocladius wygodzinskyi (n = 4) were edited
in GENEIOUS trial v.7.1.3 with a final length of 655 bp. Sequences of specimens as well as relevant collateral
information was uploaded to the BOLD. Accession codes: GLAND001-22, GLAND002-22, GLAND003-22,
GLAND004-22.
DNA barcode analysis and phylogenetic tree reconstruction
Additional 28 sequences of COX1 partial gene of Orthocladiinae species were retrieved from GenBank and BOLD
system. Analysis included the following parasitic chironomids: nine sequences of Nanocladius (Plecopteracoluthus)
shigaensis Inoue, 2015 and six of Nanocladius (Plecopteracoluthus) asiaticus Hayashi, 1998 from Japan; three of
Epoicocladius flavens Malloch, 1915 from USA; one of Epoicocladius ephemerae Kieffer, 1925 from Finland;
one of Epoicocladius ephemerae; three of two unidentified species of Epoicocladius, two of Symbiocladius (S.)
rithogenae, and two of an unidentified species of Symbiocladius from China. Barbadocladius was chosen as
outgroup due to its recognized as a sister clade of the Orthocladinae with Gondwanic distribution. It possesses
distinctive morphological traits that are unique within the subfamily. The four sequences of Symbiocladius (A.)
wygodzinskyi generated in the present study were also included in the analyses.
Thirty-two sequences were aligned by MUSCLE method with default parameters in MEGA v11 (Tamura et
al., 2021). The alignment was then manually edited by cutting primers. Our final alignment is 655 bp in length.
Phylogenetic tree was constructed using Neighbor-Joining (NJ) method and 1000 bootstrap replicates. The NJ tree
was edited in Inkscape 1.2.2. Genetic distances (inter- and intraspecific) were estimated considering Kimura 2-
parametres (Kimura, 1980) model and 1000 replicates in MEGA.
RESULTS
Symbiocladius wygodziskyi (Roback, 1965)
The morphological analysis of the larval stage of S. wygodzinskyi agrees with the character descriptions given in
Kieffer (1925), Roback (1965) and Gonser & Spies (1997) for mentum with 5–6 lateral teeth and the proximal tooth
of the mandible long and slender. On the other hand, mature larvae have curved thoracic horns, gradually narrowing
from the wide base to acute apex. Moreover, molecular analysis also supports morphological results. More detailed
results are described below.
Larva
Body length: 1.94–2.33 mm (n = 2), maximum diameter: 0.36–0.29 mm (n = 2).
Body with yellowish and slightly whitish coloration on the sides (Fig. 2A–B). Cephalic sclerotized capsule
triangular in shape, 134.9–147.7 μm (n = 2) long, with a maximum width at the base of 135.3–139.3 μm (n = 2).
Non-visible eye spots (Fig. 2D). Antenna with 5 segments, 14–18.1 μm (n = 3) long. Mandible, length: 25–28.7 μm
(n = 4), heavily sclerotized, with two internal teeth, the more distal tooth is thicker than the proximal one. Mentum
translucent not heavily sclerotized as the mandibles, wide central tooth, with 5–6 very acute lateral teeth at distal
end. Short anterior prolegs with more than 90 hooks, which present one apical tooth and several internal teeth.
Posterior prolegs with 19 hooks, these hooks do not have internal teeth.
Pupa
Mature larvae already display pupal features, including sclerotized thoracic horns. Body length: 3.15–4.10 mm (n =
2), maximum width (thorax): 1.02–1.03 mm (n = 2).
VELÁSQUEZ-RODRÍGUEZ ET AL.
52 · Zootaxa 5319 (1) © 2023 Magnolia Press
The body exhibits a gray coloration, with pale or light-colored spots mainly in the ventral and lateral region of
the thorax. (Fig. 2C). Thoracic horn curved, tapering towards the distal end until ending in a very sharp projection,
127.4–143.3 μm (n = 4) total length, base of the projection is 23.7–35.6 μm (n = 4) wide (Fig. 2E). Abdomen has
filiform spines on the tergites.
FIGURE 2. Symbiocladius wygodzinskyi, A–B, larva on a Leptophlebidae host, C, larvae in process of pupation on a
Leptophlebidae host, D cephalic capsule, E thoracic horn.
Ecological notes
Specimens were found living on nymphs of Leptophlebiidae (Ephemeroptera). Larvae in process of pupation build
a membranous sheath and were positioned from the II thoracic segment to the V abdominal segment of the host.
The position of the larva on the host can be with the head forward or backward (Fig. 2A–B). There is no indication
that Symbiocladius (A.) wygodzinskyi feeds on the hemolymph of its host. Finally, the host nymphs had very small
FINDINGS IN THE GENUS SYMBIOCLADIUS Zootaxa 5319 (1) © 2023 Magnolia Press · 53
translucent wing patches of the same size. Therefore, it was not possible to appreciate the effect of the commensal
on the development of the wing patches.
Distribution: Department of La Libertad. Province: Sánchez Carrión. Municipality: Sarín. Chusgón River
(a tributary of the Marañón River), 7°58’18.195” S, 77°57’51.962” W, m.a.s.l. Department: Apurímac. Province:
Cotabambas. Municipality: Cotabambas. Anchapillay River (tributary of the Apurímac River), 14°2’57.8106” S,
72°24’6.8148” W, 4234 m.a.s.l. Department: Áncash. Province: Huaraz. District: Independencia. Churup River
9°29’14.70” S; 77°25’58.92” W, 4183 m.a.s.l. (Fig. 1)
DNA barcode analyses
The NJ tree based on partial COX1 gene shows two well supported major clades. The first clade is composed by
species of Symbiocladius (99.5% of bootstrap support) from China and the second clade is formed by the species
Symbiocladius (Acletius) wygodziskyi, the genera Nanocladius spp. and Epoicocladius spp. and Epoicocladius spp.
(92.5% of bootstrap support) (Fig. 3). This evidences that Symbiocladius does not form a monophyletic clade for
COX1 genetree. These two clades recovered from Symbiocladius agree with the currently proposed subgenera,
Symbiocladius and Acletius, presenting a genetic distance of 24.8±2%.
The first clade of Symbiocladius includes species of the subgenus Symbioclaidus: S. (S.) rithogenae and one
not described yet, S. (S.) sp. 1XL that are genetically differentiated in 17.2±1.7% (Table. 1). On the other hand, the
second clade of Symbiocladius include the sequences generated in this study for Symbiocladius (A.) wygodziskyi,
which differ from 19.4% to 26.4% with respect to the other species of Epoicocladius, and Nanocladius (Table. 1).
TABLE 1. COX1 genetic distances estimated with Kimura 2-paremeter. Interspecific distances are shown below the
diagonal and the error estimates above the diagonal. Intraspecific variation is shown in bold in the first column.
Species Intra 1 2 3 4 5 6 7 8 9 10
1Barbadocladius sp. - 1.8 1.9 2.1 1.8 1.9 1.9 2 1.7 1.8
2Symbiocladius sp.1X 6.3 ± 1.0 19.8 1.7 2.3 2.0 2.1 2.1 2.1 1.9 1.9
3Symbiocladius (S.) rhithrogenae 018.4 17.2 2.1 1.9 2 2.1 2.1 1.9 2
4Symbiocladius wygodzinskyi 023.3 26.4 23.3 1.9 1.9 2.1 2 1.9 1.9
5Epoicocladius ephemerae 10.9 ± 1.4 19.4 24.9 22.6 20.9 1.3 1.5 1.5 1.5 1.4
6Epoicocladius sp.1XL - 19.2 23.9 19.9 19.8 11.8 1.5 1.5 1.5 1.5
7Epoicocladius flavens 1.4 ± 0.4 20.2 23.5 23.4 23.3 14.9 13.2 1.5 1.5 1.6
8Epoicocladius sp. 3 ± 0.7 19.1 23.3 22.8 20.8 15.2 13.6 14.8 1.5 1.6
9Nanocladius (P.) shigaensis 2.2 ± 0.4 15.7 21.9 20.6 19.4 15.3 13.8 14.4 14.8 1
10 Nanocladius (P.) asiaticus 0.7 ± 0.2 16.2 21.5 20.6 18.7 13.9 13 15.5 14.7 7.1
DISCUSSION
Symbiocladius (Acletius) wygodzinskyi is reported for the third time in Peru. The present record complements
the distribution of the species along the Peruvian Andes in Áncash, at 175.9 km and 742.3 km from its previous
records in La Libertad and Apurímac, respectively. Moreover, this is the first time reporting molecular data for the
species.
The first report of the species in Peru by Prat et al. (2013) detailed morphological characteristics that agree with
our specimens, except for the shorter length of the larval antennae observed in our specimens (14–18.1 μm long)
compared to the previous report (33 μm long). The position of the larvae in process of pupation and immature larvae
on the host agrees with Roback (1965), Prat et al. (2013) and Gonser & Spies (1997).
Two subgenera have been proposed for Symbiocladius based on morphology, according to Roback (1965). Both
subgenera (Symbiocladius and Acletius) are differentiated by the morphology of the empodium, tibial spurs and
claws, number of palpal segments and presence or absence of eye hairs in adults, while by the shape and number of
lateral teeth of the mentum and mandible as well as the position on the host in immature stages. Moreover, species
VELÁSQUEZ-RODRÍGUEZ ET AL.
54 · Zootaxa 5319 (1) © 2023 Magnolia Press
of these subgenera are recovered in two different groups in the NJ tree (Fig. 3) and show greater genetic divergence
(23.3% and 26.4%, see Table 1). These values are comparatively quite high considering the average of interspecific
distance of 16.2% reported among Chironomidae species by Ekrem et al. (2007) and Lin et al. (2015) and the
genetic distance between species included in this study (from 7.1% and 17.8%, see Table 1). Even more, the genera
Epoicocladius and Nanocladius exhibit less genetic differences (14.5%) than the two groups of Symbiocladius
(24.8%). Additionally, despite having a large geographical distance (USA, China and Finland), sequences among
Epoicocladius species (13.6% to 15.2%) are more similar and recovered in a single group in the NJ tree than
sequences of Symbiocladius that came from Peru and China, which are highly different (23.3% to 26.4%) and
recovered as part of different groups in the NJ tree.
FIGURE 3. Neighbor joining tree of partial COX 1 sequences (655bp) of some Orthocladinae species. Bootstrap values on
nodes. Scale is based on the number of base substitutions per site. Tags shown the GenBank or BOLD system accession
code|species|collection number|country of the record.
Although the morphological analysis suggests that the Peruvian individuals belong to the genus Symbiocladius,
the significant genetic differences observed between the sequences of specimens from Peru and China indicate the
possibility that the genus Symbiocladius may be paraphyletic. However, a definite taxonomic decision cannot be
made until additional sequences from other Symbiocladius species, supported by morphological data, are included.
Furthermore, our study confirms the presence of Symbiocladius (Acletius) wygodzinskyi in Peru, provides the
first molecular data for this particular species, and highlights the need for future research to conduct a systematic
and taxonomic revision of the genus in order to resolve the discrepancies identified in this study.
Acknowledgments
This research was supported by the National Fund for Scientific and Technological Development and Technological
Innovation from Peru (FONDECYT) under the financial scheme E035-ERANet LAC—Collaborative Research
FINDINGS IN THE GENUS SYMBIOCLADIUS Zootaxa 5319 (1) © 2023 Magnolia Press · 55
Projects (Global observatory network for freshwater biodiversity in high mountain streams—GLOBIOS), and
Contract No. 006-2019 under the financial scheme E031-2018-01-NERC (CASCADA: Cascading Impacts of
Peruvian Glacier Shrinkage on Biogeochemical Cycling and Acid Drainage in Aquatic Ecosystems—Toxin or Treat?
). We are indebted to Renzo Mera, Leonela Rojas, Álvaro Recoba, Rodrigo Moreno, Jessica García, Kimberly
Peceros, Rosa Chuchón and Jean Pierre Merino for laboratory support, and to Fiorella La Matta, Vanessa Arévalo
and Cristina Gutiérrez for help with field samplings and logistics.
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