Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 August 2023 | 15(8): 23771–23777
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.8178.15.8.23771-23777
#8178 | Received 06
September 2022 | Final received 21 May 2023 | Finally accepted 08 August 2023
Description of a new species of
the genus Anthaxia (Haplanthaxia
Reitter, 1911) from India with molecular
barcoding and phylogenetic analysis
S. Seena
1, P.P. Anand 2 & Y. Shibu Vardhanan
3
1,2,3 Biochemistry & Toxicology
Division, Department of Zoology, University of Calicut, Kozhikode, Kerala
673635, India.
1 seena.s1984@gmail.com, 2 anandpp633@gmail.com
(corresponding author), 3 svardhanan@gmail.com (corresponding
author)
1,2 These authors contributed equally
to this work.
ZooBank: urn:lsid:zoobank.org:pub:493B80E1-320B-4AB6-BE97-0D9BB178D043
Editor: Mark G. Volkovitsh,
Zoological Institute of the Russian Academy of Sciences St.Petersburg, Russia. Date of publication: 26 August 2023 (online & print)
Citation: Seena, S., P.P. Anand & Y.S. Vardhanan (2023). Description of a new species of the
genus Anthaxia (Haplanthaxia
Reitter, 1911) from India with molecular barcoding
and phylogenetic analysis. Journal of Threatened Taxa 15(8): 23771–23777. https://doi.org/10.11609/jott.8178.15.8.23771-23777
Copyright: © Seena al. 2023. 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: The present study was financially supported by UGC-SAP, Government of India (F.3-6/212 (DAP-II) dated 10.10.2012).
Competing interests:The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.
Author details: Dr. Seena, S. completed her PhD from University of Calicut and the thesis dealt with the morphology, molecular, morphometric study of Jewel beetles of Kerala, South India with special emphasis on antennal sensilla structure and light reflection mechanisms of Buprestid. P. P.
Anand doing Ph. D research (University of Calicut) on molecular aspects of mussel foot proteins. Dr. Y. Shibu Vardhanan working as associate professor in Zoology, University of Calicut. His lab focused diverse aspects such as geometric morphometrics, toxicology, Biochemistry, molecular biology, biomaterial characterization and waste management.
Author contributions: Field level collection: SS; Description: SS and PPA; molecular analysis: SS and PPA; Supervision:
YSV.
Acknowledgements: The authors are thankful to the
authorities at Department of Zoology, University of Calicut for providing
facilities. The authors also thankful to Dr. P.M. Sureshan, officer-in-charge and Dr.
P. Girish Kumar, Western Ghat Regional Centre,
Zoological Survey of India, Kozhikode, India for the constant support and
encouragement throughout this work.
Abstract: This paper deals with the
description of a new Anthaxia (subgenus Haplanthaxia Reitter,
1911) species from southern India, which belongs to the Anthaxia (H.) winkleri Obenberger, 1914 species-group: Anthaxia
(H.) keralensis sp. nov. In addition to a morphological description, we
also generated mt. COI DNA sequences and discuss the
results of a phylogenetic analysis of the new species with previously deposited
COI DNA sequences of Anthaxia spp.
In a maximum-likelihood phylogenetic analysis, the new species shared the same
hypothetical ancestor node with A. melancholica Gory,
1841 and similar molecular characteristics (~48% similarity) with A. tenella Kiesenwetter,
1858 and A. corinthia Reiche & Saulcy, 1856. More
systematic studies are required to understand the species diversity,
distribution, biology, and evolutionary significance of the Anthaxia
(H.) species groups.
Keywords: Beetle, Buprestidae,
CO1 gene, Coleoptera, molecular phylogeny, oriental
region, southern India, Western Ghats.
INTRODUCTION
Buprestidae is one of the world’s largest coleopteran
families, with nearly 15,000 extant species in 522 genera (Bellamy 2008). The
genus Anthaxia Eschscholtz, 1829, is a diversified taxon with a wide
distribution; this genus includes 697 species worldwide (Bellamy 2008; Kubáň 2016). The genus Anthaxia comprises
eight subgenera (Bílý 2019), of which Haplanthaxia Reitter,
1911 is the largest, comprising 70% of species of the genus. There are
currently 20 defined species-groups in the subgenus Haplanthaxia and
many more awaiting definitions (Bílý 2017, 2019). Due
to its worldwide distribution and the extreme morphological similarity of some
species, it is considered as the taxonomically most challenging group in Buprestidae (Bílý 2019). Anthaxiini from the Oriental region, particularly from the
Indian subcontinent, have received little attention. Southern Indian Anthaxia (H.) has not yet been
studied; in this work, we discuss the new species from Anthaxia species
group.
In addition
to the morphological description, we discuss the molecular phylogenetic
position of our new species among relative species. Due to limited sampling,
the Buprestidae group’s molecular identification,
classification, and phylogenetic analysis are not yet well developed. At
present, species identification and classification are primarily based on
morphological characteristics. Compared to other buprestid genera, Agrilus Curtis, 1825, which has received
the most attention in molecular barcoding and phylogenetic analysis. Kelnarova et al. (2018) investigated and developed the
first DNA reference library for ~ 100 Agrilus species
from the Northern Hemisphere using three mitochondrial markers: cox1-5’ (DNA
barcode fragments), cox1-3’, and rmL. Rapid
detection and taxonomic identification of buprestid species is the first step,
especially if the species is economically significant. Recently, mitochondrial
DNA-based species identification methods have become increasingly important as
a practical alternative to classical morphology-based identification (Herbert
et al. 2003; Riedel et al. 2013a, b; Ashfaq & Herbert 2016). Here, we
present the first molecular mt. CO1 barcoding
sequence of the genus Anthaxia from
India, with the first mt. CO1 phylogeny analysis of
all known Anthaxia species
available in NCBI and BOLD databases.
MATERIALS
AND METHODS
Specimens
studied here were collected with yellow pan traps from the Aralam
wildlife sanctuary (11.95050N 75.82310E, 238 m) in Kannur district, southern Western
Ghats, Kerala, India. Images were taken with a Carl Zeiss SteREO
Discovery.V20 microscope with a 6MP CCD sensor camera 506 attached and
processed with Adobe Photoshop CS8 to standardize background and remove artifacts
formed during stacking. In addition, measurements of body parts of holotype
specimen were taken with Carl Zeiss SteREO Discovery
V20 inbuild software. The holotype and paratype are deposited in the Department
of Zoology, University of Calicut (DZUC) and will be transferred to the
National Collections of Zoological Survey of India, Western Ghat
Regional Centre, Kozhikode, Kerala (ZSIK).
The body
length was measured in the middle of the body following the elytral suture (the
same for the pronotal and elytral length); width of
the body was measured at the maximum body width (usually the maximum span
between lateral pronotal margins or span between the
outer margin of humeral callosities) (Bílý 2020). The
terminology used to describe surface sculpture is based on Harris (1979).
DNA
extraction, amplification, sequencing, and phylogenetic analysis
Genomic DNA
was extracted from the thoracic leg using Nucleospin®
Tissue Kit (Macherey-Nagel) following the
manufacturer’s instructions. The extracted DNA was subjected to PCR
amplification. PCR was performed in a reaction mixture containing 6.25 µL
master mix (PCR master mix: Phire Hot Start II PCR
Master Mix, Thermofisher, Cat. No: F125S), 1.25 µL
forward and reverse primer, 1 µL extracted DNA sample and 3.25 µL water. The
total volume of the reaction mixture is 13 µL. For performing PCR mitochondrial
cytochrome c oxidase subunit 1 (CO1) amplification, we used Lep
primer (LepF1 5’ ATTCAACCAATCATAAAGATATTGG 3’ and LepR1 5’
TAAACTTCTGGATGTCCAAAAAATCA 3’) (Herbert et al. 2004; Wilson 2012). The thermal
profiles of CO1 amplification were 5 min at 95o C, 40 cycles of 10
sec at 94o C, 1 min at 52o C, and 45 sec at 72o C,
followed by a final extension of 10 min at 72o C. The purified PCR
products were sequenced at Rajiv Gandhi Centre for Biotechnology (RGCB),
Thiruvananthapuram, Kerala, India, using the dideoxy chain termination method
(Sanger & Coulson 1975). The forward and reverse strands were aligned using
Clustal W in MEGA X to ensure the sequences were
clear without any mismatches, frameshift regions, premature stop codons,
etc.
The
sequences were checked in the NCBI BLAST tool to find similar sequences in the
NCBI database. All mt. CO1 DNA sequences of Anthaxia species were retrieved from NCBI and BOLD
database and aligned in MEGA X, MUSCLE alignment method (Kumar et al. 2018),
and the aligned sequences were used for phylogeny construction analysis. To
find out the best model for phylogeny analysis, we performed maximum likelihood
fits of 24 different nucleotide substitution models. Models with the lowest BIC
scores (Bayesian information criterion) are considered to describe the
substitution pattern the best. For each model, AICc
value (Alkaline information criterion, corrected), Maximum likelihood value (InL), and the number of parameters (including branch
lengths) are also validated (Nei & Kumar 2000). A
total of 30 nucleotide sequences (including new species CO1) were used for
phylogenetic analysis. GTR+G+I (General Time Reversible model + Gamma
Distributed with Invariants Sites) model is the best model for the phylogeny
construction analysis of the genus Anthaxia
(Parameters = 67; BIC = 10045.924; AICc =
9554.572; InL = -4709.885).
Phylogenetic
relationship of taxa was analysed by using maximum
likelihood and neighbour-joining method. The
evolutionary history was inferred using the maximum likelihood method and the
General Time Reversible model (Nei & Kumar 2000).
The bootstrap consensus tree inferred from 1,000 replicates (Felsenstein 1985) is taken to represent the evolutionary
history of the taxa analysed (Felsenstein
1985). Branches corresponding to partitions reproduced in less than 50% of
bootstrap replicates are collapsed. The percentage of replicate trees in which
the associated taxa clustered together in the bootstrap test (1,000 replicates)
are shown next to the branches (Felsenstein 1985).
Initial tree(s) for the heuristic search were automatically obtained by
applying neighbour-join and BioNJ
algorithms to a matric of pairwise distances estimates using the maximum
composite likelihood (MCL) approach, then selecting the topology with superior
log likelihood value. A discrete Gama distribution was used to model
evolutionary rate differences among sites (5 categories (+G, parameter =
0.2661)). The rate variation model allowed some sites to be evolutionarily
invariable ([+l], 18.07% sites). This analysis involved 30 nucleotide
sequences (including new species CO1). Codon positions included were 1st+2nd+3rd+Noncoding.
All positions with less than 95% site coverage were eliminated, i.e., fewer
than 5% alignment gaps, missing data, and ambiguous bases were allowed at any
position (partial deletion option). There was a total of 384 positions in the
final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al.
2018).
RESULTS
Anthaxia (Haplanthaxia) winkleri
Obenberger, 1914 species group
Small to
medium-sized species (4.0–6.0 mm). The head is wide, the forehead is flat,
wide, the eyes projecting beyond head contour; upper lobe of eye more obtuse.
On the broad vertex, the inner rims of the eyes are far apart, diverging
towards vertex. Frons rather variable, from flat, slightly grooved in the
middle, to widely depressed; frontal pubescence thicker, more
sparse, rather reclined; clypeus almost flat. The forehead is always
dark copper or green in colour, as is the rest of the
body, only rarely a little lighter. The pronotum is almost twice as wide as
long, fairly flat, depressed, the posterior angles not protruding backwards at
all. It is widest in the anterior third, from there to the base and to the
anterior margin finely and weakly, equally narrowed. Anterior margin deeply bisinuate, with pronounced central lobe. The posterior
angles are rectangular. The structure of the pronotum is regular; it consists
of low cells similar to those of the head; these are half extinct, very fine,
only the central granules protrude somewhat more clearly, the walls of the
cells are almost distinct. Scutellum slightly wider than long. The elytra are
flat, without depressions, finely granulated, margins deep & wider, not
shagreened, dark coppery, slightly wider in the shoulders than pronotum, individually
tapered, and rounded at the apex. Metatibiae proportionally shorter, stronger,
inner edge usually more strongly sinuate, incised, acutely serrate, with
stronger, more acute apical spur. Aedeagus narrower, less sinuate; apex median
lobe subparallel, more angulate, acutely pointed.
TAXONOMY
Anthaxia (Haplanthaxia) keralensis sp.
nov.
(Image 1,2;
Figure 1)
urn:lsid:zoobank.org:act:AF553762-19DC-438D-8BBA-8EE282C7130D
Material
examined
Holotype: DZUC
BLAK001, male, 10.vi.2019, Aralam Wildlife Sanctuary,
Kerala, India, (11.95050N, 75.82310E, 238 m), coll:
S. Seena”.
Paratype: DZUC
BLAK002, male, same as holotype.
Measurement
(Holotype): total length 5.71 mm, the width of head 1.65 mm, length of pronotum
1.08 mm, the width of pronotum 1.89 mm, length of elytra 3.87 mm, and width of
elytra 1.92 mm.
Diagnosis: Medium-sized
(5.7 mm) (Image 2A), robust; frons, vertex and pronotum bright green metallic
with bronze lusters; elytra bronze with bright green lusters; ventral surface,
antennae and legs bronze-green metallic, metepimera
and abdominal ventrites green with bronze lusters;
pronotum with distinct deep posterolateral depressions; lateral sides of 1st
abdominal segment with tomentose spot; metatibiae
straight, with dense hispid bristles externally; entire body covered with setose, golden yellow, small erect pubescence (Image 2).
Description
of the holotype
Head
slightly wider than anterior pronotal margin; frons
convex, vertex weakly depressed, 0.5 times as wide as width of eye; frontoclypeus anteriorly slightly convex; eyes large,
narrowly reniform, slightly projecting beyond the outline of the head; inner
ocular margins parallel, feebly converging toward vertex; sculpture of head
consisting of very small, dense, polygonal cells with central grains; short
erect yellow pubescence uniformly distributed; clypeus roughly micro-sculptured
(Image 2D); antennae long, almost reaching posterior pronotal
angles when laid alongside; scape claviform, about 4 times as long as wide,
pedicel suboval, about 1.5 times as long as wide;
third antennomere triangular, about twice as long as wide, antennomeres 4–10
trapezoidal, slightly longer than wide, terminal antennomere rhomboid, twice as
long as wide.
Pronotum
weakly convex, 1.8 times as wide as long, with wide, distinct deep postero-lateral depressions; anterior margin bisinuate, posterior margin almost straight; lateral
margins widely, regularly rounded, posterior angles obtuse-angled, maximum pronotal width at midlength; pronotal sculpture consisting of a simple, fine, network of
subpolygonal cells with weakly raised borders,
slightly denser on latero-posterior areas; cell bottom strongly
micro-sculptured, with distinct central grain; bearing short, erect, golden yellow
pronotal pubescence. Scutellum small, finely micro
sculptured, pentagonal, as wide as long (Image 1A).
Elytra
regularly convex and tapering posteriorly, 2.9 times as long as wide; basal,
transverse depressions shallow, not reaching scutellum, humeral callosities
small, only weakly projecting beyond elytral outline; elytral epipleura rather wide, parallel-sided, almost reaching
elytral apex; lateral preapical serrations very fine, the apex of each elytron
broadly rounded; elytral sculpture almost homogeneous, consisting of fine,
dense, simple punctures with small erect golden pubescence; apex of elytra
weakly dentate (Image 2A, 2C).
Ventral
surface lustrous with finely ocellate sculpture, cell borders weakly raised;
abdominal ventrites almost glabrous; prosternal process wide, subparallel, with well-developed
and acute lateral angles; anal ventrite weakly
truncate apically, slightly angulate and rather strongly serrate laterally
(Image 2B). Legs long and slender, protibiae weakly
curved, meso- and metatibiae straight, with dense
hispid bristles externally (Image 2E); tarsal claws delicate, hook-shaped, not
enlarged at base.
Aedeagus
long, slender, weakly spindle-shaped, dorso-ventrally
flattened, and the median lobe sharply pointed apically (Image 2F).
Female: unknown.
Etymology: The new
species is named after the Indian state Kerala where the holotype was
collected.
Distribution: India,
Kerala State, known only from the type locality.
Differential
diagnosis: This species is similar to A. marshalli Stebbing, 1914 and A. (H.) tanjorensis Obenberger, 1938 by
size and general habitus but it distinguishes by its setose
body, uniformly distributed golden yellow short erect pubescence, tomentum on
the lateral side of 1st ventrite (Image
2C), and aedeagus shape (Image 2F). A. (H.) keralensis sp. nov. is easily
distinguished from A. (H.) tanjorensis
Obenberger, 1938 by its pronotonal
sculpture, since A. (H.) keralensis sp.
nov. has a pronotal
sculpture usually regularly polygonal on the whole pronotal
surface, but in longitudinally stretched on discal
area as in A. (H.) tanjorensis.
Molecular
phylogeny analysis
A total of
29 mt.CO1 barcoding sequences of the genus Anthaxia available
in NCBI and BOLD database. In the ML phylogenetic analysis (Figure 1), the tree
divides into two major clades, one clade containing seven species and the other
clade containing 23 species. Anthaxia
(H.) keralensis sp. nov.
(OM141594.1) is positioned in a distinct clade. A. (H.) keralensis sp. nov.
and A. melancholica diverged from
the same hypothetical ancestor node. A. (H.) keralensis sp. nov.
showed a molecular relationship (~48% of similarity) with A. melancholica, A. tenella, and A.
corinthia. The resulted molecular phylogeny
of Anthaxia has a strongly
preliminary character because all main clades and subclades have very low nodal
support. Basal clade which includes new species, is formed by representatives
of the subgenera Haplanthaxia (A.
deleta [= A. caseyi]
- A. melancholica) and Melanthaxia (A. tenella and A.
corinthia). In the same time all other species of
subgenus Melanthaxia form a
monophyletic most distant subclade (A. sepulchralis -
A. godeti). Intermediate subclades are also
mainly polyphyletic and include representatives of subgenera Anthaxia s.str., Haplanthaxia and Cratomerus.
Only basal subclade of the second clade comprises species of Haplanthaxia. It’s important to remember that
one of the factors contributing to the preliminary uncorrelated relationship of
some Anthaxia spp. was a lack of data in genebanks. For the purpose of studying the molecular
phylogenetic link among the Anthaxia, multilocus-based gene barcoding and the development of
phylogenies with extremely comparable taxa will be helpful. More molecular and
morphological systematic studies are required to understand the phylogenetic
relationship among the Anthaxia spp.
CONCLUSION
The lack of
proper revision of species from the Indian subcontinent and the high degree of
morphological variability in the A. winkleri species
group are significant impediments in assigning and describing a new species
from India. The lack of appropriate molecular barcode sequences in GenBank
databases makes mt. CO1 barcoding ineffective for
species identification at the moment. Nevertheless, we can use the barcode for
molecular phylogeny and genetic similarity analysis. A. (H.) keralensis sp. nov.
showed no close similarity with previously studied Anthaxia spp.
A multiple gene sequencing studies are required to confirm the species group belonging
of newly described species and to build the molecular phylogeny and their
evolutionary origin of the genus Anthaxia.
For
figure & images - - click here for full PDF
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