The mitochondrial genome of a leaf insect Phyllium westwoodii (Phasmatodea: Phylliidae) in Southeast Asia

Abstract

The nearly complete mitochondrial genome (mitogenome) of Phyllium westwoodii, a typical leaf mimic insect in Phasmatodea, was obtained in this study. This mitogenome is 17,222 bp in length and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and almost complete control regions. All PCGs initiate with ‘ATN’ except for NAD4L that uses ‘TTG’ as the start codon, and terminate with ‘TAA’ except for COX2 that uses a single ‘T’ residue as the stop codon. The phylogenetic analysis based on the concatenated sequences of 13 PCGs and two rRNAs shows that P. westwoodii is closer to Phyllium tibetense than Phyllium giganteum.

Full text

Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tmdn20
Mitochondrial DNA Part B
Resources
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tmdn20
The mitochondrial genome of a leaf insect Phyllium
westwoodii (Phasmatodea: Phylliidae) in Southeast
Asia
Zhiwei Dong, Jun Li, Jinwu He, Guichun Liu, Chuyang Mao, Ruoping Zhao &
Xueyan Li
To cite this article: Zhiwei Dong, Jun Li, Jinwu He, Guichun Liu, Chuyang Mao, Ruoping
Zhao & Xueyan Li (2021) The mitochondrial genome of a leaf insect Phyllium�westwoodii
(Phasmatodea: Phylliidae) in Southeast Asia, Mitochondrial DNA Part B, 6:3, 888-890, DOI:
10.1080/23802359.2021.1886014
To link to this article: https://doi.org/10.1080/23802359.2021.1886014
© 2021 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 15 Mar 2021.
Submit your article to this journal
View related articles
View Crossmark data

MITOGENOME ANNOUNCEMENT
The mitochondrial genome of a leaf insect Phyllium westwoodii (Phasmatodea:
Phylliidae) in Southeast Asia
Zhiwei Dong
a
, Jun Li
a
, Jinwu He
a,b
, Guichun Liu
a
, Chuyang Mao
a
, Ruoping Zhao
a
and
Xueyan Li
a
a
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China;
b
School of Ecology and Environment, Northwestern Polytechnical University, Xi’an, China
ABSTRACT
The nearly complete mitochondrial genome (mitogenome) of Phyllium westwoodii, a typical leaf mimic
insect in Phasmatodea, was obtained in this study. This mitogenome is 17,222 bp in length and con-
tains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs)
and almost complete control regions. All PCGs initiate with ‘ATN’except for NAD4L that uses ‘TTG’as
the start codon, and terminate with ‘TAA’except for COX2 that uses a single ‘T’residue as the stop
codon. The phylogenetic analysis based on the concatenated sequences of 13 PCGs and two rRNAs
shows that P.westwoodii is closer to Phyllium tibetense than Phyllium giganteum.
ARTICLE HISTORY
Received 16 November 2020
Accepted 2 February 2021
KEYWORDS
Mitogenome; leaf insect;
Phyllium westwoodii;
phylogenetic analysis
The Phasmatodea is known as stick and leaf insects, and cur-
rently contains three suborders, 13 families and more than
3,000 species (Zhang et al. 2011; Bradler et al. 2014). They
occur across the tropics and only a few inhabit in temperate
areas (Bradler et al. 2014). As typical representatives of
Phasmatodea, only a small percentage of extant phasmids
(78 extant species belonging to the leaf insects in the
Phylliidae) exhibits an extreme form of morphological and
behavioral leaf mimicry (Wedmann et al. 2007; Brock et al.
2020), which originated at least 47 million years (Wedmann
et al. 2007) . Thus, leaf insects are one of ideal models to
investigate the adaptive evolution of leaf mimic traits.
However, its genetic basis remains completely unknown. To
date, the only available molecular data resources for leaf
insects are two mitogenomes deposited in GenBank (Komoto
et al. 2011; Zhou et al. 2017).
In the present study, as the first step to dissect de novo
reference genome of one representative leaf insect, we used
Illumina next-generation sequencing (NGS) data to generate
the nearly complete mitogenome (with the exception por-
tions of the control region) of Phyllium westwoodii (Wood-
Mason 1875). This species is one member in Phylliidae and
mainly distribute in countries of Southeast Asia, including
India (South Andaman Island), Myanmar, China (Yunnan),
Thailand, Laos, Vietnam, Sumatra, and Singapore etc.
(Hennemann et al. 2009). Male and female adults were col-
lected from Muang Fuang, Nang Ha, Laos (10270300E;
183901200N) during June 2017 by Zhiwei Dong and local vil-
lagers, and bred in the greenhouse with host plant Rubus sp.
The voucher specimens (male: KIZ0127554, female:
KIZ0127555) are stored in Kunming Natural History Museum
of Zoology, Chinese Academy of Sciences. Their offspring (1st
instar larvae) was used to extract genomic DNA (gDNA) using
a Gentra Puregene Blood kit (Qiagen, Hilden, Germany) based
on instructions. Paired-end library (350-bp insert size) was
prepared using NEB NextV
RUltra DNA Library Prep Kit and
sequenced on Illumina HisSeq4000 (Novogene, Beijing,
China). 288 Gb total number of bases (SRA number:
SRR13336961-SRR13336964) were obtained. After low quality
reads were filtered, clean mito-reads were extracted based
on homology comparison as previously described (Tang et al.
2014). Mitogenome was assembled using SOAPdenovo-Trans
version 1.03 (kmer ¼37) (Xie et al. 2014) and subsequently
the gaps was filled using GapCloser version 1.12 (Luo et al.
2012). Gene annotation was performed by MITOS2 webserver
(http://mitos2.bioinf.uni-leipzig.de/index.py) (Bernt et al. 2013)
with manual corrections on cyclization of mitogenome
sequence, confirmation of relative position of 37 genes, and
the determination of the start and stop codes of 13 protein-
coding genes with reference to the mitogenomes (listed as
in Figure 1) of Phasmatodea deposited in GenBank.
The nearly complete mitogenome of P. westwoodii total-
izes 17,222 bp, which contain 37 typical mitochondrial genes
(13 protein-coding genes (PCGs), 22 transfer RNA genes
CONTACT Xueyan Li lixy@mail.kiz.ac.cn State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of
Sciences, #21,QingsongLu, Ciba, Kunming 650203, Yunnan, China
These authors contributed equally to this work.
ß2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
MITOCHONDRIAL DNA PART B
2021, VOL. 6, NO. 3, 888–890
https://doi.org/10.1080/23802359.2021.1886014

(tRNAs) and two ribosomal RNA genes (rRNAs)) and two
unconnected fragments of control region (GenBank accession
No. MW229063.1). The total length of all 13 PCGs is
11,131 bp with a strong bias toward A þT (76.35%), and
count 64.63% of the whole mitogenome. All PCGs initiate
with ‘ATN’(N represents A, T, G, C) except for NAD4L that
uses ‘TTG’as the start codon. Unlike the custom of terminat-
ing with ‘TAA’,COX2 stops with a single ‘T’as previously
reported (Wolstenholme 1992). The 22 tRNA genes range
from 62 bp to 71 bp in length. 21 of them have a typical clo-
ver-leaf structure except the trnS1(gct), in which the dihy-
drouridine (DHU) arm is replaced by a simple loop that is
ubiquitous in most insects (Jiang et al. 2016). The size of
large rRNA (rrnL) and small rRNA (rrnS) is 1,276 bp and
793 bp, respectively.
The mitogenomic sequences (including the nucleotide
sequencesof13PCGsandtworRNAs)ofnewlygeneratedin
this study (P. westwoodii), and previously reported from 22
Phasmatodea species (Mikheyev et al. 2017;Zhouetal.2017)
were used to reconstruct phylogenetic tree with Aposthonia
japonica and Aposthonia borneensis (Embioptera: Oligotomidae)
as outgroups. 13 PCGs and two rRNA were aligned by MEGA-X
(Kumar et al. 2018), respectively. TrimAl v1.4.rev22 (Capella-
Gutierrez et al. 2009) was used to remove unreliably aligned
sites (gt ¼0.5). The sequence matrix with 13,171 aligned
nucleotide sites were concatenated using SequenceMatrix v1. 8
(Vaidya et al. 2011) prior to phylogenetic analyses.
PartitionFinder v2.1.1 (Lanfear et al. 2017)wasruntwiceto
select best-fit partitioning schemes and models of evolution for
nucleotide (GTR þGþI). Maximum likelihood (ML) tree was
reconstructed using RAxML v8.2.10 (Stamatakis 2014)basedon
the rapid bootstrap (BS) algorithm with 1000 bootstrap repli-
cates. Our phylogenetic analysis shows that P. westwoodii is
closer to Phyllium tibetense than Phyllium giganteum (Figure 1).
Acknowledgements
The authors would thank Mr. J.C.M. Sau and other anonymous villagers
for helps in collecting and breeding samples. Our thanks are also given
to Dr. Ru Zhang and Dr. Ping Hu for discussion in data analysis. LXY
designed this study. DZW, LJ, HJW and LXY wrote this manuscript. HJW,
MCY and DZW made data analysis. DZW, LGC, and ZRP carried out
experiments.
Disclosure statement
The authors declare no conflicts of interest.
Funding
This work was funded by grants from the National Natural Science
Foundation of China [No. 32070482] and from Chinese Academy of
Sciences (CAS ‘Light of West China’) (to LXY).
ORCID
Zhiwei Dong http://orcid.org/0000-0002-5040-7904
Jun Li http://orcid.org/0000-0002-1040-0986
Jinwu He http://orcid.org/0000-0003-1681-9769
Guichun Liu http://orcid.org/0000-0002-0899-7808
Chuyang Mao http://orcid.org/0000-0003-1625-4470
Xueyan Li http://orcid.org/0000-0003-0457-7846
Data availability statement
The genome sequence data that support the findings of this study are
openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov)
under the accession no. MW229063.1. The associated BioProject, Bio-
Sample, and SRA numbers are PRJNA682332, SAMN16988089 and
SRR13336961-SRR13336964, respectively.
Figure 1. Inferred phylogenetic relationships among Phasmatodea based on the nucleotide sequence of concatenated 13 protein-coding genes (PCGs) and two
ribosomal RNA genes (rRNAs) (rrnL and rrnS) using maximum likelihood (ML) analysis. Both Aposthonia japonica and Aposthonia borneensis were used as outgroups.
The species Phyllium westwoodii in this study is highlighted in bold.
MITOCHONDRIAL DNA PART B 889

References
Bernt M, Donath A, Juhling F, Externbrink F, Florentz C, Fritzsch G, Putz J,
Middendorf M, Stadler PF. 2013. MITOS: improved de novo metazoan
mitochondrial genome annotation. Mol Phylogenet Evol. 69(2):
313–319.
Bradler S, Robertson JA, Whiting MF. 2014. A molecular phylogeny of
Phasmatodea with emphasis on Necrosciinae, the most species-rich
subfamily of stick insects. Syst Entomol. 39(2):205–222.
Brock PD, B€
uscher T, Baker E, Otte D. 2020. SF Phasmida: phasmida spe-
cies file (version 5.0, Jun 2018). In: Roskov Y, Ower G, Orrell T,
Nicolson D, Bailly N, Kirk PM, Bourgoin T, DeWalt RE, Decock W,
Nieukerken E van, Penev L, editors. Species 2000 & ITIS catalogue of
life, 2020-09-01 beta. Leiden (The Netherlands): Naturalis. ISSN 2405-
8858. Available from www.catalogueoflife.org/col.
Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for
automated alignment trimming in large-scale phylogenetic analyses.
Bioinformatics. 25(15):1972–1973.
Hennemann F, Conle O, Gottardo M, Bresseel J. 2009. On certain species
of the genus Phyllium Illiger, 1798, with proposals for an intra-generic
systematization and the descriptions of five new species from the
Philippines and Palawan (Phasmatodea: Phylliidae: Phylliinae: Phylliini).
Zootaxa. 2322(1):1–83.
Jiang P, Li H, Song F, Cai Y, Wang J, Liu J, Cai W. 2016. Duplication and
remolding of tRNA genes in the mitochondrial genome of Reduvius
tenebrosus (Hemiptera: Reduviidae). Int J Mol Sci. 17(6):951.
Komoto N, Yukuhiro K, Ueda K, Tomita S. 2011. Exploring the molecular
phylogeny of phasmids with whole mitochondrial genome sequences.
Mol Phylogenet Evol. 58(1):43–52.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular
evolutionary genetics analysis across computing platforms. Mol Biol
Evol. 35(6):1547–1549.
Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. 2017.
PartitionFinder 2: new methods for selecting partitioned models of
evolution for molecular and morphological phylogenetic analyses. Mol
Biol Evol. 34(3):772–773.
Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y,
et al. 2012. SOAPdenovo2: an empirically improved memory-efficient
short-read de novo assembler. Gigascience. 1(1):18.
Mikheyev AS, Zwick A, Magrath MJL, Grau ML, Qiu L, Su YN, Yeates D.
2017. Museum genomics confirms that the Lord Howe Island stick
insect survived extinction. Curr Biol. 27(20):3157.e4–3161.e4.
Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis
and post-analysis of large phylogenies. Bioinformatics. 30(9):
1312–1313.
Tang M, Tan M, Meng G, Yang S, Su X, Liu S, Song W, Li Y, Wu Q, Zhang
A, et al. 2014. Multiplex sequencing of pooled mitochondrial
genomes-a crucial step toward biodiversity analysis using mito-meta-
genomics. Nucleic Acids Res. 42(22):e166.
Vaidya G, Lohman DJ, Meier R. 2011. SequenceMatrix: concatenation soft-
ware for the fast assembly of multi-gene datasets with character set
and codon information. Cladistics. 27(2):171–180.
Wedmann S, Bradler S, Rust J. 2007. The first fossil leaf insect: 47 million
years of specialized cryptic morphology and behavior. Proc Natl Acad
Sci U S A. 104(2):565–569.
Wolstenholme DR. 1992. Animal mitochondrial DNA: structure and evolu-
tion. Int Rev Cytol. 141:173–216.
Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S,
et al. 2014. SOAPdenovo-Trans: de novo transcriptome assembly with
short RNA-Seq reads. Bioinformatics. 30(12):1660–1666.
Zhang Z, Hooper J, van Soest R, Pisera A, Crowther A, Tyler S, Schilling S,
Eschmeyer W, Fong J, Blackburn D, et al. 2011. Animal biodiversity: an
outline of higher-level classification and taxonomic richness. Zootaxa.
3148(1):7–237.
Zhou Z, Guan B, Chai J, Che X. 2017. Next-generation sequencing data
used to determine the mitochondrial genomes and a preliminary
phylogeny of Verophasmatodea insects. J Asia-Pac Entomol. 20(2):
713–719.
890 Z. DONG ET AL.