Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 August 2022 | 14(8): 21612–21618
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
https://doi.org/10.11609/jott.7830.14.8.21612-21618
#7830 | Received 12 January 2022 | Final
received 07 July 2022 | Finally accepted 23 July 2022
Description of the
larva of Vestalis melania
(Selys, 1873) (Odonata: Calopterygidae)
identified through DNA barcoding
Don Mark E. Guadalquiver 1, Olga M. Nuneza
2, Sharon Rose M. Tabugo 3 & Reagan Joseph T. Villanueva 4
1,2,3 Department of
Biological Sciences, College of Science and Mathematics, MSU-Iligan Institute
of Technology, Andres Bonifacio Avenue, Tibanga,
Iligan City, Philippines.
2 Biodiversity Research
Group, Premier Research Institute of Science and Mathematics, Iligan City 9200,
Philippines.
4 College of Arts and
Sciences Education, University of Mindanao, Matina,
Davao City, 8000 Philippines.
1donmark.guadalquiver@g.msuiit.edu.ph
(corresponding author), 2 olgamnuneza@yahoo.com, 3 sharonrose.tabugo@g.msuiit.edu.ph,
4 rjtvillanueva@gmail.com
Editor: Anonymity requested. Date of publication: 26 August
2022 (online & print)
Citation: Guadalquiver,
D.M.E., O.M. Nuneza, S.R.M. Tabugo
& R.J.T. Villanueva (2022). Description of the larva of Vestalis melania (Selys, 1873) (Odonata: Calopterygidae)
identified through DNA barcoding. Journal of Threatened Taxa 14(8): 21612–21618. https://doi.org/10.11609/jott.7830.14.8.21612-21618
Copyright: © Guadalquiver et al. 2022. Creative Commons
Attribution 4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: Department of Science and
Technology (DOST)
Competing interests: The authors
declare no competing interests.
Author details: Mr. Don Mark Guadalquiver is a graduate of MS
Biology and worked on biodiversity studies. He is currently pursuing his
studies in the field of medicine. Dr.
Olga Nuneza is a professor in Biology in the
Department of Biological Sciences, MSU-Iligan Institute of Technology working on
Biodiversity studies. Dr. Sharon Rose Tabugo
is also a professor in Biology in the Department of Biological Sciences,
MSU-Iligan Institute of Technology working on biodiversity, molecular
systematics and evolution. Dr. Reagan Joseph Villanueva is affiliated
with the University of Mindanao and is one of the authorities in Philippine
Odonata, a member of the IUCN Odonata Specialist Group.
Author contributions:
DMEG—conceptualization, data curation, methodology,
investigation, formal analysis, writing; OMN: supervision, methodology,
writing; SRRT—supervision, methodology, investigation, validation, writing;
RJTV—supervision, validation, writing.
Acknowledgements: The authors would
like to acknowledge the Provincial Government of Bukidnon, the City of
Malaybalay, and DENR Region X for granting the necessary permits, the Brgy. Officials of Kibalabag for
the accommodation, the DOST-ASTHRDP grant for the funding, and Mr. Jayson R. Pucot for the technical assistance and for lending
materials used in this study.
Abstract: The larva of Vestalis melania is
described and illustrated for the first time, based on specimens collected from
Malaybalay, Bukidnon, Philippines. The identity of the larva was confirmed by
matching its mitochondrial COI sequence with the adult. The larva can be
distinguished by the shape of the prementum and its
median cleft, lateral gills, and posterolateral abdominal spines. Comparison
with other known larvae in the genus is also provided. The significance of
using DNA barcoding for identifying larvae of Philippine Odonata is emphasized.
Keywords: COI sequencing,
Odonata, Philippines, Mindanao, Zygoptera
INTRODUCTION
Vestalis Selys,
1853 is a genus of the Calopterygidae family with 16
species distributed in the Oriental region (Lieftinck
1965; Paulson & Schorr 2021). Like other members of Calopterygidae,
the species thrive well in pristine habitats with good water quality (Orr
2003). In the past, the genus was subdivided into three groups, which were
treated as full genera based on neural and penile characters (May 1935). These
three are Vestalis Selys,
1853, Vestinus Kennedy, 1920, and Vestalaria May, 1953. Lieftinck
(1965) dismissed this division, stating the instability of the characters
defining Vestinus. However, molecular
and morphological data supported the resurrection of the genus name Vestalaria (Hämäläinen
2006).
Vestalis melania, a member of the
genus Vestalis, is geographically distinct for
its insular distribution and restriction in the Philippines (Lieftinck 1965). The
species is widely distributed in the country, except in Palawan, thrives mainly
in the open or partly shaded streams and rivers (Villanueva 2009). Presently,
only two of the 16 species within Vestalis
have described larvae which are V. amoena and V.
luctuosa (Ris 1912; Lieftinck 1965). Hence, in this study, the larva of V. melania was described for the first time. Larval
identity was confirmed by matching the mitochondrial COI sequence of larvae and
adults, a method increasingly utilized in Odonata (Orr & Dow 2015a,b, 2016; Steinhoff et al. 2016; Yu 2016; Wang et
al. 2017; Saetung & Boonsoong
2019). Detailed morphology of the larva was also described and compared with
other known larvae in the genus to gain more insights into its phylogenetic
position.
MATERIALS AND METHODS
Collection of
Specimens
Larval specimens were
collected from the streams of Kibalabag, Malaybalay
City, and Bukidnon. Specimens were collected through sieving substrates, leaf
debris, and water vegetation in the margins of streams or water pockets near
streams. Samples collected were preserved in 95% ethanol. All materials are
deposited in the Natural Science Museum (NSM-4293 to NSM-4296) of Mindanao
State University-Iligan Institute of Technology, Iligan City, Mindanao,
Philippines. The collection was made under the DENR wildlife gratuitous permit
no. R10-2021-27.
DNA Extraction and
Polymerase Chain Reaction
Genomic DNA was extracted
from the legs of specimens using the EZ-10 Spin Column Genomic DNA Minipreps
Kit (BioBasic, Canada). The animal DNA barcode, COI (cytochrome c
oxidase subunit I), was amplified by universal primers
(5’GCTCAACAAATCATAAAGAYATYGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAARAAYTCA-3’)
(Folmer et al. 1994). Each PCR reaction contains 30
µL of PCR master mix (Bio Basic, Inc.), 18 µL of ddH2O, 3 µL of each primer,
and 6 µL of DNA template for a total volume of 60 µL. The PCR thermal regime
consisted of pre-denaturation at 94 °C for four mins; 35 cycles of denaturation
at 94°C for 30 sec., annealing at 48.5 °C for 30 sec., and extension at 72 °C
for 90 sec.; final extension at 72 °C for seven mins; and hold for 4 °C at ∞.
PCR products were then subsequently visualized on 1.5% agarose gel (Bio-rad) using blueGel
electrophoresis system (Minipcrbio, Amyplus). PCR products were then sent to Macrogen Korea for sequencing.
DNA Barcode Analysis
The forward and
reverse COI sequences were edited using Snapgene
Viewer 5.2.5.1 (GSL Biotech; available at snapgene.com). Consensus sequences
were then generated through queries of the forward and reverse sequence in NCBI
Blast. Sequence analyses were carried out using MEGA 10 (Kumar et al. 2018).
Pairwise distances were calculated using Kimura-2-parameter model using all
sites and 1,000 bootstrap replications to determine the genetic distance
between conspecific individuals.
Imaging and
Description
Specimens were
examined and photographed using a stereo microscope with an attached digital
camera (AmScope) and a Canon EOS 60d. Illustrations
were created through an Ipad using the procreate
application (Savage Interactive, Australia), based on representative images.
Measurements were obtained through ImageJ (Schneider et al. 2012).
Terminologies for the larval morphology were based on Snodgrass (1954) and
Kumar (1973). The mandibular formula follows Watson (1955). Abdominal segments
1–10 were indicated as S1–S10.
RESULTS
The COI sequences of
all samples were amplified and sequenced successfully, producing barcodes
568–576 bp long. A maximum-likelihood tree including
11 reference sequences from Vestalis and Vestalaria (Table 1) is shown in Figure 1. Euaphaea formosa
was used as an outgroup. The adults of Vestalis
melania distinctly formed a monophyletic group
(MLB = 100) with prospect larvae. Vestalis smaragdina, a member of Vestalaria,
also had the closest relationship with V. melania,
forming a monophylum (MLB = 58). The results confirmed that the larvae
concerned here are of the same species as adults, namely V. melania.
Taxonomic Account
Vestalis melania Selys,
1873
Materials studied: Larvae: 14.x.2020, 1
male, 3 females: Kibalabag, Malaybalay City,
Bukidnon, Philippines (8.258 N, 125.172 E), 1,200 m, coll. D.M. Guadalquiver, Natural Science Museum, MSU-IIT, Iligan,
Philippines
Description: A slender zygopteran with a small head, moderately long antennae,
laterally banded thorax with long and banded legs, elongated and cylindrical
abdomen with lanceolate lamellae. Ground color of light brown but can be darker
in some individuals (Image 1).
Head: Hexagon-shaped with a
pointed snout, flattened above, with light banding, pointed & pigmented postocular lobe, and eyes longer than wide when dorsally
viewed. Antennae (Figure 2a) seven-segmented excluding extra joint after
segment 1, tapered from base to apex, with robust segment one almost twice as
long as segments 2–7. Prementum (Figure 2b) elongated
with the distal end expanding at angles 110 to 1250 wide. Median lobes (Ligula) clefted
roundly and with deepness 0.36 of the prementum,
serrated on the outside, and containing a pair of setae. Labial palp robust,
the inner lateral margin serrated with two sizes of teeth, and with three
strong, long, and incurved distal teeth, of which the middle one is the
longest; movable hook very long and robust with two setae on its base. Maxilla
(Figure 2c) is twice as long as wide; galeo-laccinia
with seven teeth: four long in the dorsal area and three short in the ventral area, and with numerous hair-like projections.
Palpus is
two-segmented, with a small basal segment and distal segment that is
banana-shaped but pointed, as long as galeo-laccinia,
and covered in numerous hair-like projections. Mandibles (Figure 2d,e) with the
formula L 1’1234 0 a(m1,2,3,4,5-7)b/ R 1’1234 y a. Left mandible
with five incisors and molar crest with 5–7 fine cusps; right mandible with
five incisors, an extra tooth, and a single mandible.
Thorax: Marked with strong
bandings in the lateral area extending from the pronotum up to the dorsal
region of synthorax. Prothorax smaller than head and synthorax. Pronotum hexagonal with a protuberance at the
mediolateral proximities. Wing pads reaching the proximal margin of S4. Legs
long and with two dark bands in femur and tibia and progressively longer from
pro- to meta-thorax. Tibia longer than femur; tarsi three-segmented and covered
with dense hair.
Abdomen: Long & slender
and covered with dark pigmentation, amount varying between specimens, but less
pigmented on the median region. Lateral spines on S9 and S10, with S10 spine
more prominent (Figure 3a,b). Male gonapophyses
protruding from the middle of S9, small and conical with black pigmentations in
the upper lateral area (Figure 3a). Female inner gonapophyses
large and extending from proximal margin of S9 to distal margin of S10; outer
part protruding from middle of S9 to distal margin of S10, with distal region
slight pointed upward (Figure 3b). Male cerci small and budlike; female cerci
more pointed and slightly longer than male ones. Caudal gills are all
lamellate, long, lanceolate shaped but blunt-tipped, and bearing some fine
setae-like spines along margins. The lateral caudal gill (Figure 3d) is longer
than the middle gill, with a prominent midrib and light to dark pigmentation
covering the entire median region; banding manifests only in the lateral edges.
Middle caudal gill (Figure 3c) with full banded pigmentation, translucent, and
visible median venations.
Microhabitat and
Behavior
Larvae were found in
an unshaded, narrow, montane stream with a sandy substrate and dense marginal
and submerged vegetation (Image 2). Larvae are abundant where they are found
and were found clinging and scooped along with submerged vegetation. Adults of
V. melania and Euphaea
amphicyana were also abundant in the area.
DISCUSSION
The only described
species within the genus Vestalis are V. luctuosa (Ris 1912; Lieftinck 1965) and V. amoena
(Lieftinck 1965). Comparison of the larval morphology
of V. melania from descriptions of these
species shows that V. melania is different in
several aspects. Vestalis melania shows stronger banding in the pronotum up to
the thorax; round median cleft in the prementum
compared to angular base, and sharp broadening in the anterior region of the prementum compared with gradual broadening in the other
species.
The lateral gills
also differ with V. luctuosa and V. amoena, in terms of pigmentation. In V. melania, pigmentation was concentrated and full in the
central region, and the banding is observable only in the edges, giving it an
appearance of having ‘white’ spots in the borders. In contrast, the lateral
gills of V. luctuosa are less pigmented and
show dark spots in the edges (Ris 1912), whereas V.
amoena does not show much pigmentation and has a
truncated shape (Lieftinck 1965) (Table 2).
The posterolateral
spines in the abdomen of V. melania were also
remarkable, being prominent in S9–10. In V. amoena
and V. luctuosa (Lieftinck
1965), a small spine is also present in S10, but it is unclear if it is also
present in S9.
Overall, the larval
characteristics of V. melania are different in
terms of stronger banding in the pronotum and thorax, characters in the prementum, lateral caudal gill, and posterolateral spines
in the abdomen.
This study
demonstrates once again the usefulness of DNA barcoding in matching the larvae
with the adult. This method can be utilized to gain larval knowledge of endemic
Philippine Odonata, especially endemic genus like Risiocnemis.
As the marker COI has been proven helpful in differentiating many Philippine
damselfly species (Casas et al. 2018), it can be effectively utilized to match
most larvae and adults of the same species. Caution, however, should be
observed in using COI genes for some species groups, as the gene may not be
well-differentiated in some closely related species such as Philippine Drepanosticta species (Casas et al. 2018). Another
example is the Vestalis gracilis
and V. apicalis used in this study
which showed no divergence (MLB = 100). Hence, other gene targets should be
utilized as well, such as the nuclear ribosomal genes and the internal
transcribed spacers.
Conclusion
This study describes
for the first time the larvae of Vestalis melania. The mitochondrial COI sequence successfully
matched the V. melania larvae with its adults
and confirmed its identity, in congruence with their sympatric relationship.
The larva of V. melania is highly
similar to previously described congener species but different in terms of
stronger banding in the pronotum and thorax, characters in the prementum, lateral caudal gill, and posterolateral spines
in the abdomen. The larval morphology of the V. melania
supports the unity within the genus Vestalis
and the separate genus status of Vestalaria.
It is recommended that larvae of other Vestalis
species be further studied and DNA barcoding, should be incorporated to gain
more larval knowledge of endemic Philippine Odonata. Because of the limitations
of the COI marker in closely related species, it is also recommended that other
gene targets and relevant data should be used for support.
Table 1. Specimen
data of COI sequence used in the analysis.
|
Species |
Data Source |
ID/AN |
Locality |
Date |
Collector |
|
Vestalis melania |
BOLD |
SKODO086-15 |
Tagbina, Surigao del Sur, Philippines |
27.ii.2015 |
H. Cahilog |
|
Vestalis melania |
BOLD |
SKODO047-15 |
Tboli, South Cotabato, Philippines |
22.viii.2015 |
H. Cahilog |
|
Vestalis melania* |
This study |
KMBPH011/ NSM-4293 |
Kibalabag, Malaybalay City, Philippines |
14.x.2020 |
D.M. Guadalquiver |
|
Vestalis melania* |
This study |
KMBPH015/ NSM-4294 |
Kibalabag, Malaybalay City, Philippines |
14.x.2020 |
D.M. Guadalquiver |
|
Vestalis melania * |
This study |
KMBP016/ NSM-4295 |
Kibalabag, Malaybalay City, Philippines |
14.x.2020 |
D.M. Guadalquiver |
|
Vestalis amethystina |
NCBI |
MG885367.1 |
Singapore |
|
|
|
Vestalis amabilis |
NCBI |
KF369567.1 |
Sarawak, Malaysia |
01.01.2010 |
J. Teo |
|
Vestalis amoena |
NCBI |
MG885091 |
Singapore |
|
|
|
Vestalis amoena |
NCBI |
MG885368.1 |
Singapore |
|
|
|
Vestalis apicalis |
NCBI |
KU510326.1 |
India |
|
|
|
Vestalis apicalis |
NCBI |
MN255519.1 |
India |
|
|
|
Vestalis gracilis |
NCBI |
KX503058 |
India |
|
|
|
Vestalis gracilis |
NCBI |
MN387793.1 |
India |
|
|
|
Vestalis smaragdina |
NCBI |
KF369577.1 |
|
|
|
|
Euphaea formosa |
BOLD |
GBMHO2948-19 |
Taiwan |
|
|
For
figures & images - - click here for full PDF
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