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Revisiting the evolution of Ostrinia moths with phylogenomics (Pyraloidea: Crambidae: Pyraustinae)


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Reconstructing a robust phylogenetic framework is key to understanding the ecology and evolution of many economically important taxa. The crambid moth genus Ostrinia contains multiple agricultural pests, and its classification and phylogeny has remained controversial because of the paucity of characters and the lack of clear morphological boundaries for its species. To address these issues, we inferred a molecular phylogeny of Ostrinia using a phylogenomic dataset containing 498 loci and 115 197 nucleotide sites and examined whether traditional morphological characters corroborate our molecular results. Our results strongly support the monophyly of one of the Ostrinia species groups but surprisingly do not support the monophyly of the other two. Based on the extensive morphological examination and broadly representative taxon sampling of the phylogenomic analyses, we propose a revised classification of the genus, defined by three species groups (Ostrinia nubilalis species group, Ostrinia obumbratalis species group, and Ostrinia penitalis species group), which differs from the traditional classification of Mutuura & Munroe (1970). Morphological and molecular evidence reveal the presence of a new North American species, Ostrinia multispinosa Yang sp.n., closely related to O. obumbratalis. Our analyses indicate that the Ostrinia ancestral larval host preference was for dicots, and that O. nubilalis (European corn borer) and Ostrinia furnacalis (Asian corn borer) independently evolved a preference for feeding on monocots (i.e., maize). Males of a few Ostrinia species have enlarged, grooved midtibiae with brush organs that are known to attract females to increase mating success during courtship, which may represent a derived condition. Our study provides a strong evolutionary framework for this agriculturally important insect lineage.
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Systematic Entomology (2021), 46, 827– 838
Revisiting the evolution of Ostrinia moths with
phylogenomics (Pyraloidea: Crambidae: Pyraustinae)
1Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Northwest A&F University, Yangling,
China, 2Entomological Museum, College of Plant Protection, Northwest A&F University, Yangling, China, 3McGuire Center for
Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL, U.S.A., 4Entomology and
Nematology Department, University of Florida, Gainesville, FL, U.S.A. and 5Agriculture and Agri-Food Canada, Ottawa Research
and Development Centre, Ottawa, ON, Canada
Abstract. Reconstructing a robust phylogenetic framework is key to understanding
the ecology and evolution of many economically important taxa. The crambid moth
genus Ostrinia contains multiple agricultural pests, and its classication and phylogeny
has remained controversial because of the paucity of characters and the lack of
clear morphological boundaries for its species. To address these issues, we inferred a
molecular phylogeny of Ostrinia using a phylogenomic dataset containing 498 loci and
115 197 nucleotide sites and examined whether traditional morphological characters
corroborate our molecular results. Our results strongly support the monophyly of one
of the Ostrinia species groups but surprisingly do not support the monophyly of the
other two. Based on the extensive morphological examination and broadly representative
taxon sampling of the phylogenomic analyses, we propose a revised classication of
the genus, dened by three species groups (Ostrinia nubilalis species group, Ostrinia
obumbratalis species group, and Ostrinia penitalis species group), which differs from
the traditional classication of Mutuura & Munroe (1970). Morphological and molecular
evidence reveal the presence of a new North American species, Ostrinia multispinosa
Yang sp.n., closely related to O.obumbratalis. Our analyses indicate that the Ostrinia
ancestral larval host preference was for dicots, and that O.nubilalis (European corn
borer) and Ostrinia furnacalis (Asian corn borer) independently evolved a preference
for feeding on monocots (i.e., maize). Males of a few Ostrinia species have enlarged,
grooved midtibiae with brush organs that are known to attract females to increase mating
success during courtship, which may represent a derived condition. Our study provides
a strong evolutionary framework for this agriculturally important insect lineage.
The genus Ostrinia Hübner, 1825, with 23 species and 35
described subspecies worldwide, has been the subject of exten-
sive study (Mutuura & Munroe, 1970; Ohno, 2003; Frolov
et al., 2007; Leraut, 2012) owing to the inclusion of several
major agricultural pests, such as the European corn borer
(Ostrinia nubilalis (Hübner, 1796)), the Asian corn borer
Correspondence: Zhaofu Yang, Key Laboratory of Plant Protec-
tion Resources and Pest Management, Ministry of Education, College
of Plant Protection, Northwest A&F University, 3 Taicheng Road, Yan-
gling, 712100, Shaanxi, China. E-mail:
(Ostrinia furnacalis (Guenée, 1854)) and the Adzuki bean
borer (Ostrinia scapulalis (Walker, 1859)). European corn
borer is one of the most devastating pests of maize (Zea mays
L.) and, in the eastern United States, it has been responsi-
ble for yield losses exceeding $1 billion annually (Mason
et al., 1996). The high rate of Asian corn borer infestation
causes heavy damage in corn and other crops in south-east Asia
and Australia, with 10–30% corn yield reduction in China and
the Philippines (Zhou et al., 1995; Yorobe & Quicoy, 2006),
while Adzuki bean borer is a dicot-feeding pest of hops
(Humulus lupulus, Cannabaceae), hemp (Cannabis sativa,
Cannabaceae) and legumes (Vigna sp., Fabaceae), and it occurs
© 2021 The Royal Entomological Society 827
828 Z. Yang et al.
in sympatry with both maize-feeding species across Eurasia
(Frolov et al., 2007).
Despite containing some of the most devastating insect pest
species worldwide, the classication of Ostrinia remains largely
untested. Species boundaries, phylogenetic relationships within
and among species groups, and the validity of subgroups remain
unclear (Kim et al., 1999; Frolov et al., 2007, 2012; Lassance
et al., 2013). The classication that has been generally accepted
until now was rst proposed by Mutuura & Munroe (1970),
and it was based on differences in morphology of the male
genitalia and male midtibia. These authors divided the genus
into three species groups: the ‘rst species group’ consists of
a single North American species, Ostrinia penitalis (Grote),
and is characterized by a dorsally trid juxta and unarmed
sacculus. The ‘second species group’ includes nine species,
geographically ranging from North America to Africa, Siberia
and East Asia, which possess an uncus that is either simple or
bid and a sacculus with dorsal spines. The ‘third species group’
consists of 10 species with a trilobate uncus and dorsally spined
sacculus, and with distribution limited to Europe, northwestern
Africa, Asia and neighboring islands. They further subdivided
the third group into three subgroups based on differences in the
morphology of the male midtibia.
Although the classication of Mutuura & Munroe (1970) has
widely been adopted, evidence suggests that the classication
may need to be changed. For example, Frolov (1981, 1984)
used genetic crossing experiments by examining representatives
of the three subgroups of the ‘third species group’ (Ostrinia
narynensis,O.nubilalis and O.scapulalis) to show that over-
all size of the male midtibia (small, medium or large) may be a
polymorphic trait with phenotypic variation, implying that the
division of subgroups based on male midtibial morphology is
unreliable. Frolov et al. (2007) proposed that Ostrinia orien-
talis Mutuura & Munroe, 1970 and O.narynensis Mutuura &
Munroe, 1970, which were originally placed in the rst subgroup
and the second subgroup by Mutuura and Munroe, respectively,
be synonymized with O.scapulalis (Walker, 1859) in the third
subgroup. From a taxonomic standpoint, Ostrinia remains one
of the most problematic groups in terms of species delineation
within Pyraustinae.
There have been a few phylogenetic studies conducted on
Ostrinia, but all have their limitations on either gene or taxon
sampling. Based on partial mitochondrial COII gene fragments
from eight representatives of two groups, Kim et al. (1999)
supported that the ‘second species group’ is sister to the ‘third
species group’. The sister-group relationship recovered between
the ‘second’ and the ‘third’ species groups was later supported
by Yang et al. (2011) based on a single-locus mitochondrial
COI analysis, and by Lassance et al. (2013) who performed a
multilocus analysis using COI and four nuclear genes (EF-1𝛼,
MDH,IDH,RpS5). However, the phylogenetic relationships
of the genus Ostrinia were not condently resolved in these
studies due to limited gene and taxon sampling, especially in
the ‘rst species group’. The study of Zhou et al. (2020), based
on mitogenomes, showed that Ostrinia palustralis,whichwas
classied as part of Mutuura and Munroe’s second species
group, may be the sister lineage to the remaining two species
groups. However, the study of Zhou et al. (2020) included
only six of the 23 described Ostrinia species. Thus, a full
understanding of the phylogenetic relationships could not be
An important aspect of the evolution of Ostrinia is their
larval host plant associations. European corn borer and Asian
corn borer preferentially feed on maize, but Adzuki bean borer
and the remaining Ostrinia species mostly feed on dicots
(Frolov et al., 2007, 2012). This has led to the hypothesis
that the ancestral condition of Ostrinia larval feeding diet was
a dicot, and that host preferences shifted to monocots over
time (Alexandre et al., 2013; Bourguet et al., 2014). Resolving
phylogenetic relationships of Ostrinia is critical in order to
understand how these pest species evolved their ability to
become severe crop pests. An evolutionary analysis of larval
host plants in Ostrinia has yet to be formally conducted.
Here, we use anchored hybrid enrichment (AHE) of 498 loci
and close to two-thirds of the known Ostrinia species to resolve
its phylogenetic relationships, revise its classication and exam-
ine the evolution of larval host plant feeding. Specically, we
assemble AHE data for a phylogenomic analysis of Ostrinia in
order to (i) test the monophyly of the three species groups rec-
ognized by Mutuura & Munroe (1970); (ii) clarify whether the
‘rst species group’ is sister to a clade containing the other two
groups; (iii) understand the evolution of host plant associations
and morphological characters used to dene species groups and
Materials and methods
Taxon and gene sampling
We recovered DNA from 15 species Ostrinia (Table S1),
representing 65.2% of all described species. No DNA was
recovered from the samples of Ostrinia kurentzovi Mutuura
& Munroe, 1970; O.narynensis Mutuura & Munroe, 1970;
Ostrinia sanguinealis (Warren, 1892). Sequences of Ostrinia
dorsivittata (Moore, 1888) and Ostrinia erythrialis (Hamp-
son, 1913) were excluded due to high incidence of potential
cross-contamination. Four species such as Ostrinia avari-
alis Amsel, 1970; Ostrinia maysalis Leraut, 2012; Ostrinia
ovalipennis Ohno, 2003; Ostrinia putzufangensis Mutuura &
Munroe, 1970 could not be obtained for DNA sequencing.
Eight outgroups were sampled, including six newly sequenced
in this study and two from previous studies: seven from the
family Crambidae (six Pyraustinae and one Spilomelinae) and
one from the family Pyralidae (Bazinet et al., 2013; St Laurent
et al., 2018). Samples were dissected for identication based
on male genital features and compared with material deposited
in the Canadian National Collection of Insects, Arachnids and
Nematodes (CNC), as this collection has particularly strong
representation of the genus Ostrinia, including 43 types deter-
mined by specialists such as E. G. Munroe and A. Mutuura.
Genitalia preparation and photography followed Landry (2007)
and Yang et al. (2012), whereas genital terminology followed
Kristensen (2003). Tissue samples for DNA extraction are
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
Revisiting the evolution of Ostrinia moths 829
preserved at the McGuire Center for Lepidoptera and Bio-
diversity, Florida Museum of Natural History, Gainesville,
Florida, U.S.A. and the Entomological Museum, Northwest
A&F University, Yangling, Shaanxi, China.
AHE, DNA extraction, sequencing and alignments
We used AHE phylogenomics to resolve relationships of
Ostrinia. AHE uses DNA probes designed to capture hun-
dreds of informative orthologous loci throughout the genome
(Lemmon et al., 2012). Across Lepidoptera, it has shown great
promise to resolve both shallow- and deep-level relationships
among species (Hamilton et al., 2016; Breinholt et al., 2018;
Johns et al., 2018; St Laurent et al., 2018; Espeland et al., 2019;
Kawahara et al., 2019; Carvalho et al., 2020; St Laurent
et al., 2020).
Genomic DNA was extracted from whole abdomens, before
removing the genitalia for dissection, using the OmniPrep
Genomic DNA Extraction Kit (G-Biosciences, St. Louis, MO,
U.S.A.). DNA concentration was quantied using a Qubit 2.0
uorometer and dsDNA BR Assay Kit (Invitrogen, Thermo
Fisher Scientic, Carlsbad, CA, U.S.A.). The LEP1 Agilent
Custom SureSelect probe set was used to target 855 loci across
Lepidoptera (Breinholt et al., 2018). Pooled library preparation,
hybridization enrichment and Illumina HiSeq 2500 sequencing
(PE150) were carried out by RAPiD Genomics (Gainesville, FL,
Data were processed following the pipeline described in
Breinholt et al. (2018). Briey, raw Illumina reads were
ltered for quality and trimmed with  ! v. 0.4.0
(Krueger, 2015). Loci were assembled from matched reads pass-
ing quality ltering using Bombyx mori reference sequences
and iterative baited assembly (; Breinholt et al. 2018),
which employs  v. 7.0 (Edgar, 2010) and  v.
2014-12-01 (Chang et al., 2015). Orthologous and single-copy
probe regions were determined by mapping reads to the B.mori
genome. Sequencing contamination was identied and removed
using  (Camacho et al., 2009) and the custom python
script, contamination_ (Breinholt et al., 2018). The
resulting probe regions were aligned with  v.7.245 (Katoh
& Standley, 2013) and any remaining isoforms were collapsed
into 50% consensus sequences using FASconCAT-G_v1.02
with the parameters ‘-c -c -c -o -s’ (Kück & Longo, 2014).
 v.1.18.1 was used to check probe regions for any
stop codons, indels and alignment errors and to correct reading
frames (Larsson, 2014). Targeted loci that were successfully
captured and were present in at least 12 of the 15 ingroup taxa
(80%) were concatenated into a single aligned dataset with
F-G v1.02 and used for phylogenomic analyses. The
raw data for newly generated AHE sequences, the concatenated
nucleotide alignment (Data S1) and the individual locus align-
ments are provided as supplementary materials on  (doi:
10.5061/dryad.wwpzgmsj5). The raw sequence data that
support the ndings of this study are available in GenBank
of the National Center for Biotechnology Information at (Bioproject PRJNA722535).
The associated SRA data accession numbers are provided in
Table S1.
The dataset was partitioned by gene and codon position (Data
S2). To obtain the most likely tree, this partitioning scheme
was used for 100 tree searches in - v. 1.5 (Nguyen
et al., 2015). Substitution model selection and merging of par-
titions (Data S3) were done in - with the ‘MFP +merge’
option (Kalyaanamoorthy et al., 2017). Nodal support of the
  (ML) tree was estimated with 1000 ultra-
fast bootstrap replicates (; Minh et al., 2013) and
1000 replicates of the SH-like approximate likelihood ratio test
(-; Guindon et al., 2010), and the ‘bnni’ option was
used to reduce the risk of overestimating branch supports due
to severe model violations. When discussing branch support, we
refer to ‘strong support’ as relationships that have both 
95 and - 80.
Coalescent-based species-tree methods
To account for possible effects of independent gene histories
and incomplete lineage sorting, species tree estimation was per-
formed under the multi-species coalescent model in -III
v. 5.5 (Zhang et al., 2018) from each individual gene. This
method uses gene trees as input and attempts to optimize
the species tree as the topology with the largest number of
induced quartet trees from the gene tree pool. Each maximum
likelihood gene tree was generated using the default settings
in -. The single-locus datasets were not partitioned by
codon position, and a single substitution model was selected
in each individual analysis by implementing 
(Kalyaanamoorthy et al., 2017) in -. One likelihood
search was done on each single-locus dataset; in all analyses,
- performed at least 100 iterations before completing the
tree search. The resulting gene trees were subsequently input
into , which was run using default settings. Support
values on the  topology (Data S4) are given as local
posterior probabilities (LPP; Sayyari & Mirarab, 2016).
Character evolution
Ancestral state reconstructions (ASRs) were performed on
the best tree (Data S5) from the  analysis. To study the
evolution of Ostrinia larval host plant associations, we obtained
host plant records from published Ostrinia literature (Mutuura
& Munroe, 1970; Ishikawa et al., 1999) and scored these data
as two binary characters (Table S2). For the rst ASR host
plant analysis, we scored species as either feeding on only
dicots or feeding on both dicots and monocots; there are no
Ostrinia known to feed only on monocots. Since species that
feed on both monocots and dicots often have a preference for
one over the other, a second analysis was performed in which
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
830 Z. Yang et al.
Fig. 1. Maximum likelihood tree of Ostrinia estimated in - using the anchored hybrid enrichment dataset. Support values are /-.
Nodes of importance are numbered in bold. The ancestral state reconstructions (ASRs) of larval host plant associations are represented as pies on
ingroup nodes. The ASRs of the male midtibiae are indicated with capital letters following tip labels as follows: (L) enlarged, (S) small.
each species was scored as preferring either monocots or dicots.
We distinguish between these two ASR analyses in later parts of
the text by referring to the former as ‘host association’ and the
latter as ‘host preference’. Host records for four species, Ostrinia
marginalis,Ostrinia multispinosa,Ostrinia peregrinalis and
Ostrinia quadripunctalis, are unknown and those species were
treated as having missing data. We also performed ASRs to
study the evolution of four morphological characters that are
historically important for Ostrinia classication: size of the male
midtibia, shape of the uncus, presence of spines on the sacculus
and shape of the juxta (Table S2, Figs. 1 and 2).
All ASR analyses were conducted using stochastic character
mapping, with the ‘make.simmap’ command in the  package
 v06-99 (Revell, 2012). The ‘SYM’ option was used
to treat forward and reverse character state transitions as having
equal rates. One thousand stochastic maps were generated for
each ASR analysis.
Sequence capture and phylogenetic results
AHE captured 830 out of 855 loci from 15 species of Ostrinia.
We selected all loci (498/830) that were present at least12 out of
15 ingroups, resulting in a concatenated total length of 115 197
nucleotide sites and an average locus length of 231bp. All of
these loci were also represented by at least 3 out of 8 outgroups,
for a total of at least 17 species per locus.
Both  (Fig. 1) and  (Fig. S1) analyses of the
concatenated dataset recovered the genus Ostrinia as mono-
phyletic with strong bootstrap support (Node 1 in Fig. 1,
 =100/- =100; LPP =100) and as the sister
group to the Pyraustinae outgroups. Within Ostrinia,the
analyses strongly supported the denition of three monophyletic
species groups (Nodes 2, 6, 8;  =100/- =100;
 =99/- =97;  =100/- =100).
The Ostrinia obumbratalis species group (Clade I), con-
sisting of four taxa from Mutuura & Munroe’s ‘second
species group’, was recovered as sister to other Ostrinia
in both the  (LPP =100) and  trees (Node 2,
 =100/- =100). The O.penitalis species group
(Clade II) currently includes O.penitalis, the only member of
the ‘rst species group’ sensu Mutuura & Munroe (1970) and
Ostrinia latipennis (formerly of the ‘second species group’)
with strong nodal support in both the  (LPP =98)
and  trees (Node 6;  =99/- =97). The
clade consists of O.quadripunctalis +(Ostrinia kasmirica +all
members of the ‘third species group’), dened here as the
O.nubilalis species group (Clade III), was recovered as a
well-supported monophyletic group in the genus with strong
nodal support in both the  (LPP =100) and  trees
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
Revisiting the evolution of Ostrinia moths 831
Fig. 2. Male genital characters of Ostrinia spp.
(Node 8;  =100/- =100). Topological conicts
between the  and  trees occurred among relationships
within Ostrinia where relationships were characterized by low
branch support from both methods.
The ‘second species group’ recognized by Mutuura &
Munroe (1970) was recovered as paraphyletic in the 
and  analyses based on our phylogenomic data. Two
members (O.kasmirica,O.quadripunctalis) of the ‘second
species group’ were grouped with the O.nubilalis species
group (Clade III). In addition, one of the sampled taxa in
our dataset, originally recognized as O.obumbratalis PS1
in Yang et al. (2016), is now believed to be a new species,
O.multispinosa sp.n., that is sister to O.obumbratalis (the
‘second species group’) in both the  (LPP =100) and
 trees (Node 4,  =100/- =100). It was
previously shown to have signicant COI barcode divergence
compared to O.obumbratalis (Yang et al., 2016). In the present
study, we found additional morphological differences to justify
treating O.multispinosa as distinct from O.obumbratalis (see
Classication section).
The ‘third species group’ with trilobed uncus as dened by
Mutuura & Munroe (1970) was recovered as monophyletic with
strong nodal support in both the  (LPP =100) and 
trees (Node 10,  =100/- =100), which is consis-
tent with previous studies based on morphological and molecu-
lar data. In contrast, all three subgroups recognized by Mutuura
& Munroe (1970) based on male midtibia differences were
not recovered as monophyletic in either the  or the 
trees. These relationships were poorly supported in the 
tree. In contrast, some branches in the  tree received strong
support, which largely agree with previous DNA-based stud-
ies. Despite the relationships between the taxa not being fully
resolved (e.g., Nodes 12 and 13,  <95/- <80),
O.furnacalis (the ‘rst subgroup’) was sister to Ostrinia zealis
(the ‘third subgroup’) and O.orientalis (the ‘rst subgroup’) was
sister to O.scapulalis (the ‘third subgroup’) with high nodal
support (Node 11,  =100/- =60.0; Node 14,
 =99/- =95.0) in our  tree. The lowest support
value was found at Node 13. Ostrinia zaguliaevi, a species with a
strongly dilated midtibia and massive scales in the groove placed
within the ‘third subgroup’ by Mutuura & Munroe (1970), was
sister to all remaining members of the ‘third species group’ but
with low nodal support (Node 12,  =71/- =41.0)
in our  tree. These results suggested that the subgroup divi-
sions of Mutuura & Munroe (1970) are not supported. Two
important pests, O.furnacalis and O.nubilalis, were not sister
taxa, suggesting that similarities in phenotype, ecological niche
and host plant (maize) may be the result of convergence. Fur-
thermore, the position of O.scapulalis was congruent with other
studies that showed that polymorphism of male midtibia mor-
phology may lead to improper species groupings.
Character evolution
Our ASR analysis of Ostrinia larval host plant associations
(Node 1 in Figs 1, S2) showed a 52% probability that the
ancestral Ostrinia was associated only with dicots, only slightly
greater than the 48% probability of association with both dicots
and monocots. This margin was too small to conclusively
state that dicot association is the true ancestral state, but if it
was true, the results throughout the rest of the tree (Fig. S2)
would indicate at least three separate shifts to association with
both dicots and monocots, and three reversals to dicots only
(O.kasmirica,O.latipennis and O.orientalis). Host data were
missing for O.marginalis,O.multispinosa,O.peregrinalis and
O.quadripunctalis. Although the abovementioned analysis of
general host associations did not condently resolve the status
of the ancestral node, the subsequent ASR analysis that was
restricted to each species’ preferred host (Fig. S3) was much
more conclusive, showing a 95% probability that the ancestral
larval host preference of Ostrinia was a dicot.
The ASR analysis of male midtibia size (Fig. S4) showed
a 97% probability that the ancestral state was ‘small’, with
separate shifts to ‘enlarged’ midtibiae for three species:
O.scapulalis,O.zaguliaevi and O.zealis. The ASR analysis
of uncus shape (Fig. S5) showed an over 99.9% probability
that the ancestral state was ‘simple’ in the genus, with one
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
832 Z. Yang et al.
independent shift to ‘bifurcated’ in O.marginalis, and a single
shift to ‘trilobed’ in the ‘third species group’. The ASR analysis
of the sacculus (Fig. S6) showed an over 99.9% probability
that the ancestral Ostrinia sacculus was spined, with loss of
spines in O.penitalis. The ASR analysis of the juxta (Fig. S7)
showed a 52% probability that the ancestral state was ‘two
V-shaped sclerites’, with a loss of anterior sclerites in the
O.obumbratalis species group (Clade I) and a reversal to the
ancestral condition in O.peregrinalis. In the clade comprising
all the other species, there was an independent loss of anterior
sclerites in O.palustralis, and a single shift to the ‘trid’ state
in O.penitalis.
Phylogenetic relationships
Our phylogenetic analyses recover Ostrinia as monophyletic
with strong support in both  and  topologies, which
is consistent with a recent study that recovered the genus as
reciprocally monophyletic based on mitogenomic data (Zhou
et al., 2020). Since only six other genera of the diverse subfamily
Pyraustinae are included as outgroups, further phylogenomic
studies with broader taxon sampling are needed to unequivocally
demonstrate the monophyly of Ostrinia. But this is beyond the
scope of the present study.
In the  analyses, three major well-supported clades are
recognized within Ostrinia.TheO.obumbratalis species group
(Clade I) is recovered as sister to the rest of Ostrinia.TheO.
penitalis species group (Clade II) and the O.nubilalis species
group (Clade III) are both strongly supported monophyletic and
sister to each other. The morphology-based classication and
phylogenetic relationships of the genus, proposed by Mutuura
& Munroe (1970), are uncorroborated by our results. The
monophyly of the ‘rst’ and the ‘second’ species groups of
Mutuura & Munroe (1970) is not supported. In contrast, the
‘third species group’ is recovered as monophyletic with strong
support, although the relationships among the members within
the species group are not well resolved due to the rapid
radiation of the ‘third species group’. Further investigation
of this species group radiation will be helpful to shed more
light on the evolutionary history of Ostrinia. Our ndings
conrm that a classication of Ostrinia species based primarily
on male genitalia and midtibia features is problematic, as
was also revealed by other studies using mitochondrial and
nuclear makers (Kim et al., 1999; Yang et al., 2011; Lassance
et al., 2013; Zhou et al., 2020).
In our analyses, the O.obumbratalis species group (Clade I)
consists of three New World species (O.marginalis,O.multi-
spinosa sp.n.,O.obumbratalis) and Siberian O.peregrinalis,
was recovered as sister to the remainder of the genus, and the
relationships within this clade are well resolved with strong
nodal support for all nodes. In other words, these species may
constitute the most basal elements of the genus, which is incon-
sistent with previous studies based on morphological, multilocus
and mitogenomic data. Within this species group, O.marginalis
and O.peregrinalis are distributed in the boreal regions of
North America and the northwestern Palaearctic, respectively,
and represent a vicariant species pair. The dark colouration and
reduced eyes in size adapted to diurnal activity in paludicolous
habitats are likely synapomorphies for both species. Interest-
ingly, these two species differ in several characters of the male
genitalia such as the sacculus, juxta and most strikingly the
uncus (bid in O.marginalis, simple rounded in O.peregri-
nalis). Genetic evidence suggests that they are closely related
to each other despite their allopatry and appreciable difference
in male genitalia. Thus, phylogenetic relationships inferred from
conspicuous morphological criteria alone, even those involving
internal morphology, could be misleading.
It is worth noting that O.peregrinalis is represented in
our dataset by a pinned museum specimen collected over
180 years ago. This may be the oldest specimen from which
molecular data have been successfully obtained using AHE.
However, signicantly fewer loci were captured from this spec-
imen compared to others in the analysis. Its placement in the
O.obumbratalis species group is strongly supported in both
trees (Figs 1, S1), but future analyses should be conducted with
better quality specimens of O.peregrinalis that have less missing
data. Regardless, our success at extracting DNA from such an
old specimen is another example of the value that traditionally
curated museum collections provide to the scientic community.
The O.penitalis species group (Clade II) includes an allopatric
species pair, O.latipennis and O.penitalis, which are consis-
tently recovered as sister to each other with strong support in
our analyses. The Nearctic O.penitalis, the only representa-
tive of the ‘rst species group’ of Mutuura & Munroe (1970)
and thought to be one of the earliest branching lineages in the
genus, is not supported as sister to all other Ostrinia species.
Results from the present study support the hypothesis of Zhou
et al. (2020), who proposed that O.penitalis is not sister to all
other Ostrinia species. The other species O.latipennis, known
in Eastern Palaearctic and Oriental regions, is part of Mutuura
& Munroe’s ‘second species group’ and our results contradict
Mutuura & Munroe’s hypothesis on the phylogenetic placement
of this species.
For other members of the ‘second species group’, we nd
inconsistencies in the placement of O.palustralis,whichis
either sister to the O.nubilalis species group (Clade III) in the
 tree or sister to the O.penitalis species group (Clade II) in the
 analysis (Fig. S1). Further investigation of the placement
of O.palustralis, which is not dened in this study, should be the
focus of future work. In addition, two former members of the
‘second species group’, O.kasmirica and O.quadripunctalis,
are here assigned to the O.nubilalis species group, contradicting
previous studies that found alternative placements of these
species based on morphological and limited mitochondrial DNA
sequences. Therefore, our analyses strongly support that the
‘second species group’ is paraphyletic, rather than monophyletic
as proposed by Mutuura & Munroe (1970).
The O.nubilalis species group (Clade III) is the most diverse
species group in the genus, including O.quadripunctalis +
(O.kasmirica +all members of the ‘third species group’).
We nd several well-supported sister relationships among the
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
Revisiting the evolution of Ostrinia moths 833
investigated taxa of the clade with strong support. For the
monophyletic ‘third species group’, our results do not support
the monophyly of the ‘rst subgroup’ and the ‘third subgroup’
as all sampled members of these two subgroups were highly
mixed. This result suggests that the relationships among species
revealed in the present study conrmed that the male midtibia
used in Mutuura & Munroe’s (1970) classication may not the
convincing criterion to dene subgroups (Frolov et al., 2007).
Additionally, our results show that O.zaguliaevi of the ‘third
subgroup’ constitutes the basal branch of the ‘third species
group’ and is sister to the remaining species. We conrm that O.
orientalis is closely related to O.scapulalis, and that both are
valid species that are characterized by morphological, biologi-
cal and genetic differences revealed in many studies (Ishikawa
et al., 1999; Fu et al., 2004; Yang et al. 2008, Yang et al., 2011;
Yang & Zhang, 2011), which disagrees with the treatment by
Frolov et al. (2007) who considered them synonymous. No
representatives of the ‘second subgroup’, namely O.kurentzovi
and O.narynensis, were available for the present study; there-
fore, the monophyly of the subgroup needs verication and its
sister-group relationship remains unclear. The taxonomic status
of these two species and other species missing in the present
study could not be veried and require further sampling. Thus,
the incongruence of phylogeny between morphological and
molecular data needs further investigation by searching for
convincing synapomorphies and new characters for dening
the species groups and subgroups based on more comprehen-
sive taxon sampling. Additional sampling efforts would be
helpful to clarify the status of O.scapulalis and its closest
Character evolution
The most commonly recorded larval host plant association
of Ostrinia is with dicots, including the plant families Aster-
aceae, Fabaceae, Gingiberaceae, Moraceae, Polygonaceae, and
Solanaceae (Ishikawa et al., 1999). Although our analysis of
general host plant associations was not conclusive (Fig. S2), our
results show that the ancestral larval host preference of Ostrinia
is most likely a dicot (Fig. S3) and the majority of species
retained most of their ancestral host preference, which is in line
with the hypotheses proposed by Mally et al. (2019) and Léger
et al. (2021) that eudicotyledons are the ancestral host plants
of the ‘basal lineages’ (sensu Léger et al.) of Pyraustinae (i.e.,
Portentomorphini, Euclastini, Tetridia). However, a few species,
such as the two major polyphagous pests (European corn borer
and Asian corn borer) shifted their primary host plants to mono-
cots (e.g., Poaceae; Figs S2, S3). This result implies that these
two morphologically indistinguishable species likely diverged
recently in complete allopatry and shifted their host preference
to maize, supporting the hypothesis that this may have occurred
when its cultivation spread 500years ago in Europe and East
Asia (Gay, 1999; Coates et al., 2005; Malausa et al., 2007;
Calcagno et al., 2017). According to Léger et al. (2021), shifts
to monocots in the genus Ostrinia and other Pyraustinae are sec-
ondarily derived; this is corroborated by our results.
Our ASR analysis of male midtibia size (Fig. S4) shows
that the ancestral state was likely small and lacked male brush
organs, with three separate shifts to larger midtibiae with brush
organs. These brush organs are thought to be used in courtship,
where the male displays its brushes and emitting volatile
chemicals as female attractants to increase mating success
(Mallet, 1984; Birch et al., 1990; Wagner, 1991). A similar
pattern is found in some unrelated ‘lower’ Lepidoptera, such
as Hepialidae (Wagner, 1991). The calling behavior of males
attracting females may represent a derived condition restricted
to a few taxa in the ‘third species group’.
Male genitalia are often the most informative set of characters
to dene taxa and relationships across Lepidoptera, particularly
at the species level (Okagaki et al., 1955; Kristensen, 2003). Our
ASR analysis of the uncus (Fig. S5), a structure that has the func-
tion to grasp the female during copulation (Ogata et al., 1957;
Solis & Metz, 2011), indicates that the common ancestor of the
genus had a ‘simple’ uncus, whereas ‘bid’ and ‘trilobed’ unci
may represent character reversals rather than retained plesiomor-
phies. Our ASR analysis of the juxta (Fig. S7) is essentially
inconclusive, with two character states both found to have near
50% chances of being the ancestral state of the genus. The shape
of the juxta is perhaps a more rapidly evolving character rela-
tive to other aspects of Ostrinia genital morphology; this would
be consistent with the results of a previous study in which the
common ancestor of the basal Pyraustinae lineages (sensu Léger
et al., 2021) is characterized by three different states (Mally
et al. 2019). In O.penitalis, a unique ‘trid’ juxta with a long,
mid-dorsal lanceolate process is a secondary specialization,
which may be analogous to the anchor-shaped juxtal process in
Anania hortulata (Yang & Landry, 2019). Our ASR analysis of
the sacculus (Fig. S6) indicates that the ancestor of the genus
had a spined sacculus, with a secondary loss in O.penitalis.
Overall, our ndings show that Mutuura & Munroe’s (1970)
classication does not fully hold true. We therefore provide a
new classication of Ostrinia based on our phylogenetic results.
Revised classication of Ostrinia
Based on the extensive morphological examination and
broadly representative taxon sampling of the phylogenomic
results, we propose a new classication of Ostrinia,dened
by three species groups corresponding to Clades I, II and
III (Fig. 1), which differs signicantly in species group and
subgroup composition from those proposed by Mutuura &
Munroe (1970). Our results show that the ‘rst’ and the
‘second’ species groups of Mutuura & Munroe were not recov-
ered as monophyletic, whereas the ‘third species group’ was
recovered as monophyletic.
Ostrinia obumbratalis species group (Clade I in Fig. 1): This
species group consists of four species in our tree (O.marginalis,
O.multispinosa sp.n.,O.obumbratalis and O.peregrinalis).
The following male genitalia traits may be synapomorphic
for this species group: phallus with a single large cornutus,
juxta without anterior sclerites (except for O.peregrinalis
with two juxtal arms). Within this species group, we describe
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
834 Z. Yang et al.
a new species, O.multispinosa, which is closely related to
O.obumbratalis. Moreover, O.peregrinalis is found in Siberia
and eastern Europe, whereas the other three species are Nearc-
tic. A fth species, the African O.erythrialis, should be placed
in this group based on male genital features.
Ostrinia penitalis species group (Clade II in Fig. 1): This
species group consists of two species in our tree (O.latipennis,
O.penitalis). The tongue-shaped uncus of the male genitalia
may be a synapomorphy for these species. The number of
cornuti in the phallus differs: O.penitalis has a single large
cornutus, like the species in the O.obumbratalis species group,
whereas O.latipennis has two cornuti, a state shared with the
O.nubilalis species group. O.penitalis occurs in the Nearctic
and the Neotropics, whereas O.latipennis occurs in Eastern
Palaearctic and Oriental regions. O.ovalipennis, reported from
Japan, is likely to belong in this species group based on the
similarity of the male genitalia.
Ostrinia nubilalis species group (Clade III in Fig. 1): This is
the most speciose of the three groups, containing 60.1% of all
Ostrinia species. In our tree, it is represented by O.kasmirica,
O.quadripunctalis and members of the ‘third species group’
sensu Mutuura & Munroe (1970). Diagnostic characters include
having two cornuti in the phallus and a V-shaped juxta with both
anterior and posterior arms. The ‘third species group’ is recov-
ered as a well-supported monophylum of this species group, and
it is dened by having a trilobed uncus. However, the further
subdivision by Mutuura & Munroe (1970) into three subgroups
based on male midtibia is not supported. Species within the O.
nubilalis species group are distributed all over the world without
a clear distributional pattern following phylogenetic relation-
ships. Although we could not include the following species
in our analysis, O.dorsivittata,O.kurentzovi,O.maysalis,O.
narynensis,O.putzufangensis, they likely belong in the trilobed
group within this species group (Node 10, Fig. 1). Ostrinia
sanguinealis was considered as the member of the ‘second
species group’ by Mutuura & Munroe (1970). Given some
morphological traits shared with the member of the O.nubilalis
species group, for example, juxta with two V-shaped sclerites
and phallus with two cornuti, we believe that this species should
be placed in this species group regardless of its unique strongly
bid uncus (an autapomorphic trait). This species seems closely
related to O.kasmirica, which is characterized by a weakly
bid uncus and a small anterior juxtal arm.
Ostrinia multispinosa Yang s p.n ov. (Fig. 3A– D)
http://ZooBank LSID:
Type material examined. Holotype, , United States: Florida,
Putnam County, nr. Interlachen, Hwy 310 between Hwys 315
& 19, N 29.76233W 81.84347, 19 m, 16.VI.2006, P. Hebert,
K. Pickthorn, J. deWaard, 2 UV traps (CNC) [white, printed].
Labelled, BOLD sample ID and process ID: 06-FLOR-0419,
LOFLA419-06. [yellow, printed]. Genitalia slide number:
BIOZY00076. |BIOZY00076 [pale green, printed]; ‘HOLO-
TYPE|Ostrinia multispinosa|Yang 2020| by ZF Yang 2020’
[red, partly printed, partly handwritten]. Paratypes, 1,2:1,
United States: Florida, Putnam County, Etoniah Creek State
Forest, Preservation site, N 29.73920W 81.84355,36m,
17.VI.2006, P. Hebert, K. Pickthorn, J. deWaard, 3 UV traps
(CNC) [white, printed]. Labelled, BOLD sample ID and pro-
cess ID: 06-FLOR-0845, LOFLA845-06 [yellow, printed]; 1,
United States: North Carolina, Craven County, Croatan National
Forest, Swamp forest Powerline Junction, W. side Sunset Dr.,
Havelock, 5.VII.2005, J. Bolling Sullivan, UV-light trap (CNC)
[white, printed]. Labelled, BOLD sample ID and process ID:
06-NCCC-658, LNC658-06 [yellow, printed]. Genitalia slide
number: BIOZY00077. |BIOZY00077 [pale green, printed];
1, United States: North Carolina, National Forest, Swamp
Forest E. Sunset Drive, Havelok. N 34.52018W 76.56409,
8.VIII.2005, J. Bolling Sullivan, UV trap (USNM) [white,
printed]. BOLD sample ID and process ID: 06-NCCC-864,
LNC864-06 [yellow, printed]. The paratypes are provisionally
held in the CNC, Ottawa, pending agreement with the CBG
regarding their nal deposition.
Diagnosis. This species is very similar to O.obumbratalis in
external appearance, but the forewing transverse markings and
dentate subterminal band are somewhat less dened. The main
differences are in the male genitalia: in O.multispinosa,the
uncus is apically truncated, the sacculus has four spines, and the
length ratio of spined to unspined saccular areas is ca. 1.0; in O.
obumbratalis, the uncus is rounded, the sacculus has 1– 3 spines,
and ratio of spined to unspined saccular areas is less than 1.0.
Description. Head with frons smoothly scaled, pale brown-
ish yellow. Vertex whitish buff, roughly scaled. Labial pal-
pus off-white ventrally, dark brown dorsally, rst and second
palpomeres oblique, third palpomere hidden at apex of sec-
ond palpomere, porrect, fuscous, pointed apically. Maxillary
palpus fairly prominent, dark brown. Haustellum base covered
with whitish buff scales. Antenna thickened in male, basal seg-
ment large, yellowish buff dorsally, sensory surface expanded,
densely pilose; antenna of female more slender, liform. Tho-
rax whitish buff dorsally, white laterally (below wings). Tegula
fulvous, suffused with fuscous scales basally. Legs whitish buff,
suffused with fuscous scales. Forewing slender; apex rather
angulate, termen evenly curved to tornus; ground color above
pale buff, suffused with fulvous scales at costa; basal area fus-
cous; antemedial spot and discocellular spot fulvous; postmedial
fascia and subterminal band fulvous; cilia whitish buff; discal
cell half length of wing. Hindwing broadly triangular; ground
color and postmedial and subterminal bands as on forewing,
cilia white; discal cell less than half as long as wing. Abdomen
whitish buff, each segment white-banded posteriorly.
Forewing length. Female, 12.09 ±0.45 mm (n=2); male,
10.71 ±0.23 mm (n=2).
Male genitalia. Tegumen trapezoidal. Uncus tongue-shaped,
somewhat wider than long, basal part broadened, truncate
distally, laterally and distally with very few setae. Juxta with
posterior sclerites V-shaped; anterior sclerites absent. Vinculum
ventrally of moderate width, triangular. Valva rather slender;
costal margin widened in middle section, without a tuft of
specialized scales near apex; cucullus distally narrowed; apex
rounded; ventral margin slightly rounded. Sella boot-shaped, its
dorsal lobe bearing 12–13 dorsally projected, at setae with
comblike apices; its ventral part bearing 11– 12 strong spines
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
Revisiting the evolution of Ostrinia moths 835
Fig. 3. Ostrinia multispinosa sp.nov. (A) Holotype male (USA: Florida, specimen 06-FLOR-0419, forewing length =9.2mm). (B) Paratype female
(USA: North Carolina, specimen 06-NCCC-864, forewing length =12.5 mm). (C) Male genitalia (holotype genitalia slide BIOZY00076, CNC). (D)
Female genitalia (paratype genitalia slide BIOZY00077, CNC).
beyond ventral and basal margin. Sacculus about half width of
valva in mid-section, length ratio of spined to unspined saccular
part ca. 1.0; sclerotized ridge with four inwardly directed spines.
Phallus about four times as long as wide and tapered distally;
a single large cornutus with rounded tip, equal in length to
diameter of phallus, somewhat sinuous basally.
Female genitalia. Ovipositor shorter than Segment 8, with
densely setose lobes. Posterior apophysis short, bent. Eighth ter-
gite triangular, slightly wrinkled dorsally; anterior margin ven-
trad of apophysis somewhat excurved, with irregular edge. Ante-
rior apophysis about as long as Segment 8, with strengthening
bar at approximately halfway to the tip. Sterigma somewhat
triangular, enlarged anteroventrally, strongly concave dorsally
at base. Antrum cylindrical, sclerotized for a short distance;
a collar-like sclerite around ostium, then a cystoid tube for a
short distance. Inception of ductus seminalis on anterior end.
Ductus bursae about 4/5 the length of corpus bursae, membra-
nous, straight. Corpus bursae membranous, oval in outline, with
a slender rhomboidal signum, its anterior and posterior extrem-
ities extended into spinelike processes. Appendix bursae small,
membranous, oblong.
Barcode: A 658bp barcode was obtained from the holo-
type (BOLD sample ID and process ID: 06-FLOR-0419,
LOFLA419-06). Six autapomorphies shown in underlined bold
italics are located at nucleotide positions 38 T (thymidine), 364
C (cytosine), 368 A (adenine), 427 C (cytosine), 533 C (cyto-
sine), 601 C (cytosine), which are diagnostic substitutions from
other members of Ostrinia.
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
836 Z. Yang et al.
Host plant. Unknown.
Distribution. Known only from the southeastern United
States (Florida, North Carolina). It is generally sympatric with
O.obumbratalis in Florida and North Carolina. However, the
new species may occur primarily in scrub and coastal forest
based on its distributional range.
Etymology. The specic name is derived from the Latin words
multi (many) and spinosus (with spines), in reference to the
sacculus bearing four spines.
Supporting Information
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
Data S1. Concatenated nucleotide supermatrix used for ML
phylogenetic analysis.
Data S2. List of gene boundaries within the supermatrix used
as input for merging partitions with IQ-TREE.
Data S3. Best scheme for merging partitions, as determined
by ModelFinder in IQ-TREE.
Data S4. ASTRAL tree of Ostrinia in Newick format used
to generate Fig. S1.
Data S5. Maximum likelihood tree in Newick format used
to generate Fig. 1.
Figure S1. ASTRAL tree of Ostrinia. Support values are
local posterior probabilities.
Figure S2. Ancestral state reconstruction of the evolution of
larval host plant associations (black =dicots; red =monocots
Figure S3. Ancestral state reconstruction of the evolu-
tion of primary larval host preference (black =monocots;
red =dicots).
Figure S4. Ancestral state reconstruction of the evolution of
the male midtibia (black =enlarged; red =small).
Figure S5. Ancestral state reconstruction of the evolu-
tion of the uncus (black =simple; red =bifurcated;
green =trilobed).
Figure S6. Ancestral state reconstruction of the evolution of
the sacculus (black =spined; red =unarmed).
Figure S7. Ancestral state reconstruction of the evolution
of the juxta (black =trid; red =anterior sclerites of
juxta absent; green =anterior and posterior sclerites both
Tabl e S 1 Specimens sampled and included in the present
study for molecular analysis.
Tabl e S 2. Morphological character state matrix.
We are grateful to Leif Aarvik, Paul D. N. Hebert, Houhun Li,
Marko Mutanen, Gregory R. Pohl and Bo Wikström for pro-
viding freshly collected specimens. We thank Torbjørn Ekrem,
Daniel Handeld, Axel Hausmann, Derek Sikes and Patrick
Strutzenberger for permission to access their BOLD data.
Zhaofu Yang would like to thank many colleagues including
Jaret C. Daniels, John B. Heppner, Deborah M. Lott, Jacque-
line Y. Miller, Andrei Sourakov and Keith Willmott for assis-
tance during his visit to the McGuire Center for Lepidoptera and
Biodiversity. Special thanks to James E. Hayden for construc-
tive suggestions and his help to locate specimens and genitalia
slides in the MGCL collection. Lisa Bartels and Connor Lee
assisted Jean-François Landry with locating specimens and gen-
italia slides and databasing of Ostrinia in the CNC. We thank
Nan Zhou for assistance with photography of the genitalia at
NWAFU. This study was supported by the National Natural Sci-
ence Foundation of China (31772508) and NSF DBI #1349345
and DEB 1557007 to Akito Y. Kawahara. The authors acknowl-
edge University of Florida Research Computing for provid-
ing computational resources and support that contributed to the
research results reported in this publication. The authors declare
that there are no conicts of interest.
Author Contributions
Conceived the study: Zhaofu Yang, Akito Y. Kawahara. Con-
ceived and performed molecular analyses: Zhaofu Yang,
David Plotkin, Caroline Storer. Obtained morphological data:
Zhaofu Yang, Jean-François Landry. Wrote and/or edited the
manuscript: Zhaofu Yang, Jean-François Landry, David Plotkin,
Caroline Storer, Akito Y. Kawahara. Prepared illustrations:
Zhaofu Yang.
Data availability statement
NCBI Sequence Read Archive accession numbers are provided
in Table S1. The raw data for newly generated AHE sequences,
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
Revisiting the evolution of Ostrinia moths 837
the concatenated nucleotide alignment, and the individual locus
alignments are provided as supplementary materials that are
available on DRYAD (doi:10.5061/dryad.wwpzgmsj5).
Alexandre, H., Ponsard, S., Bourguet, D., Vitalis, R., Audiot, P.,
Cros-Arteil, S. & Streiff, R. (2013) When history repeats itself:
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Accepted 19 April 2021
© 2021 The Royal Entomological Society, Systematic Entomology,46, 827– 838
... Given that closely related species are very similar in morphology and appearance, species identification is extremely difficult in Lepidoptera based only on external morphological characteristics, posing problems of accuracy and speed [33][34][35]. We firstly assembled a reference DNA barcode library for the two subfamilies Pyraustinae and Spilomelinae in Crambidae to confirm species identification, and adopted four commonly used methods (Stevens' method, Pagel's method, Rohde's method, and the cross-species method) to test for the impact of the Rapoport effect on species range size [13,[36][37][38]. ...
... All sampled specimens of Pyraustinae and Spilomelinae were identified and compared with material chosen by specialist Zhaofu Yang in the Entomological Museum, Northwest A&F University, Yangling, China, based on their external morphology [25,43] and particularly considering the internal structures of female and male genitalia. The following method of genitalia slide preparation was followed Yang et al. [35]. The abdomen was removed from the dried specimens and boiled in 5-10% NaOH for 5-10 min. ...
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Rapoport’s rule proposes that a species’ range size increases with the increase in a gradient (such as latitude, altitude or water depth). However, altitudinal distributions and Rapoport’s rule have rarely been tested for Asian Lepidoptera. Pyraustinae and Spilomelinae (Lepidoptera: Crambidae) are extremely diverse in temperate Asia, including on Mount Taibai, which is considered a hotspot area for studying the vertical distribution patterns of insect species. Based on the investigation of altitudinal distribution data with identification by using both DNA barcoding and the morphological classification of Pyraustinae and Spilomelinae, this paper determines the altitudinal gradient pattern for these two subfamilies on the north slope of Mount Taibai, and provides a test of the universality of Rapoport’s rule in Lepidoptera by using four methods, including Stevens’ method, Pagel’s method, Rohde’s method, and the cross-species method. Our results show that the alpha diversity of Pyraustinae and Spilomelinae both decrease with rising altitude. By contrast, the species’ ranges increase with rising altitude. Three of the four methods used to test Rapoport’s rule yielded positive results, while Rohde’s results show a unimodal distribution model and do not support Rapoport’s rule. Our findings fill the research gap on the elevational diversity of Lepidoptera in temperate Asia.
... These two corn borers both belong to the O. nubilalis species group (trilobed uncus of male genitalia, which is a structure derived from the 10th abdominal tergite to grasp the female during copulation; see Yang et al., 2021: 830, Clade III in Figure 1), one of the most evolutionarily and ecologically interesting but taxonomically difficult groups in Lepidoptera. The species group includes 10 species and 23 subspecies worldwide (Frolov et al., 2007;Mutuura & Munroe, 1970;Yang et al., 2021). Incongruence between molecular phylogenetic relationships and the traditional classification of Ostrinia has been puzzling for a long time, leading to a number of members including ACB and ECB being morphologically indistinguishable and making accurate species identification extremely difficult (Hoshizaki et al., 2008;Kim et al., 1999;Mutuura & Munroe, 1970;Wang et al., 2017;Yang et al., 2021). ...
... The species group includes 10 species and 23 subspecies worldwide (Frolov et al., 2007;Mutuura & Munroe, 1970;Yang et al., 2021). Incongruence between molecular phylogenetic relationships and the traditional classification of Ostrinia has been puzzling for a long time, leading to a number of members including ACB and ECB being morphologically indistinguishable and making accurate species identification extremely difficult (Hoshizaki et al., 2008;Kim et al., 1999;Mutuura & Munroe, 1970;Wang et al., 2017;Yang et al., 2021). On the other hand, this species complex is a good example to understand biogeographic patterns and evolutionary histories owing to their distributional complexity of the world. ...
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Tianshan Mountains provide a model for studying biological evolution and speciation. Here we assess the evolutionary history of Ostrinia furnacalis (ACB) and Ostrinia nubilalis (ECB), which are sympatric in the Yili River Valley in Xinjiang, China.Our study is based on the historical gene flow analyses of two species by using three mitochondrial DNA (mtDNA, COI, COII, Cytb) and four nuclear DNA (nuDNA, EF-1α, Wingless, RPS5, CAD) markers obtained from representatives of HC (Huocheng), YN (Yining), XY (Xinyuan), and MNS (Manasi).Our results reveal that there is an asymmetrical gene flow pattern between the four populations. The population migratory pathways between these different populations show inflow into HC and YN, outflow from XY, and that MNS maintained a flow balance. Bayesian divergence time dating based on the COI gene suggests that the genetic divergence between the two species in this area may have occurred in Holocene at 0.008 Mya. Neutrality tests (Tajima's D, Fu's F s), and mismatch distribution test results suggest that population expansion events may not have occurred in the recent past. The demographic history and gene flow pattern between ACB and ECB may follow the "mountain isolation" hypothesis. The ML and BI trees of the mtDNA haplotype dataset show that ECB haplotypes are grouped together in a distinct clade and are clearly separate from ACB haplotypes. However, the geographical pattern of haplotype distribution is less clear for both ACB and ECB, supporting that there has been frequent gene flow among the geographic populations in the Tianshan Mountains.These findings indicate that the Tianshan Mountains are less likely a barrier to gene flow of the two species.
... Since both topologies are strongly supported in our results, we could not infer the relationship among taxa in this subgroup. Based on the morphology of male adults, Hattori & Mutuura (1987) Kim et al. 1999;Lassance et al. 2013;Luo et al. 2021;Yang et al. 2021), and our results support this as well. ...
Two of the largest subfamilies of Crambidae, Pyraustinae and Spilomelinae, have 341 species recorded in Japan, but their natural relationships are poorly understood. We reconstructed a molecular phylogeny based on one mitochondrial (COI) and three nuclear genes (CAD, EF1-α, RpS5) for 129 species in 69 genera of Spilomelinae and 62 species in 27 genera of Pyraustinae from Japan. The concatenated data was analyzed by maximum likelihood and Bayesian inference methods. Our results recovered most tribes recognized in a previous study, but Pyraustini is paraphyletic as it includes Portentomorphini, and the paraphyletic Nomophilini comprise Trichaeini. The species-rich Spilomelinae genera Patania, Nacoleia, Glyphodes , and Syllepte are polyphyletic and need thorough revision. In Pyraustinae, a “Monocotyledon-clade” and Anania sensu lato were recognized for the first time. Based on phylogenetic results and morphological investigations, we propose six taxonomic changes, including the transfer of Acropentias from Spilomelinae to Lathrotelinae and Mabra from Spilomelinae to Pyraustinae. Fourteen unplaced genera of Spilomelinae and two of Pyraustinae are newly assigned to tribes.
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Substantial differentiation was found between Ostrinia populations, adapted to feed on dicotyledonous and monocotyledonous host plants, which results not only in oviposition and larval survival differences but also in formation of ethological premating sex isolation mechanisms. Two strategies are surmised in warmer and colder areas, correspondingly: wide range of host plant species in combination with strict developmental stages of the plant, and alternatively, few host plant are infested during almost all the stages of their development, Inside these strategies, tactics are plastic. They are activated by the sensory stimuli, such as temperature, humidity and odorants. The tactic of dispersal flight before mating could be beneficial when the host plant is abundant, but mating before the flight is a better choice under the situation of sparse cornfields. There are still multiple questions to address for clear understanding of Ostrinia behavior and evolution.
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Crambidae is a group of moths with more than 10,000 species occurring worldwide that evolved diverse morphological and ecological habits. They can be best recognized by morphological characters of the adult tympanal organ and larval chaetotaxy. We present the first molecular phylogeny of Crambidae including all subfamilies and most tribes. We use available molecular data from two previous studies, and published transcriptomes and genomes, compiling ten genes totalling 11,247 bp. Up to eight genes are sequenced for thirty-nine additional taxa, with Cathariinae, Cybalomiinae and Linostinae sampled for the first time. Maximum-likelihood and Bayesian analyses recover topologies mostly agreeing with those of previous studies, with several groupings showing better support. Cathariinae syn. n. and Cybalomiinae syn. n. are recovered as ingroup of Glaphyriinae and are consequently synonymized with the latter. Linostinae are either sister to Glaphyriinae or sister to the ‘CAMMSS clade’. Lathrotelinae are recovered monophyletic and as sister to Musotiminae in the Bayesian analysis. Hoploscopinae stat. n. are recovered as sister to ((Crambinae + Erupinae stat. n.) + (Scopariinae + Heliothelinae s. str.)). Evolution of host-plant preferences is discussed.
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To understand mitogenome characteristics and reveal phylogenetic relationships of the genus Ostrinia, including several notorious pests of great importance for crops, we sequenced the complete mitogenomes of four species: Ostrinia furnacalis (Guenée, 1854), Ostrinia nubilalis (Hübner, 1796), Ostrinia scapulalis (Walker, 1859) and Ostrinia zealis (Guenée, 1854). Results indicate that the four mitogenomes—O. furnacalis, O. nubilalis, O. scapulalis, and O. zealis—are 15,245, 15,248, 15,311, and 15,208 bp in size, respectively. All four mitogenomes are comprised of 37 encoded genes and a control region. All 13 protein-coding genes (PCGs) initiate with ATN and terminate with TAN, with the exception of cox1 that starts with CGA, and cox1, cox2, and nad5 that terminate with an incomplete codon T. All transfer RNA genes (tRNAs) present the typical clover-leaf secondary structure except for the trnS1 (AGN) gene. There are some conserved structural elements in the control region. Our analyses indicate that nad6 and atp6 exhibit higher evolution rates compared to other PCGs. Phylogenetic analyses based on mitogenomes using both maximum likelihood (ML) and Bayesian inference (BI) methods revealed the relationship (O. palustralis + (O. penitalis + (O. zealis + (O. furnacalis + (O. nubilalis + O. scapulalis))))) within Ostrinia.
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Butterflies and moths (Lepidoptera) are one of the major super-radiations of insects, comprising nearly 160,000 described extant species. As herbivores, pollinators, and prey, Lepidoptera play a fundamental role in almost every terrestrial ecosystem. Lepidoptera are also indicators of environmental change and serve as models for research on mimicry and genetics. They have been central to the development of coevolutionary hypotheses, such as butterflies with flowering plants and moths' evolutionary arms race with echolocat-ing bats. However, these hypotheses have not been rigorously tested, because a robust lepidopteran phylogeny and timing of evolutionary novelties are lacking. To address these issues, we inferred a comprehensive phylogeny of Lepidoptera, using the largest data-set assembled for the order (2,098 orthologous protein-coding genes from transcriptomes of 186 species, representing nearly all super-families), and dated it with carefully evaluated synapomorphy-based fossils. The oldest members of the Lepidoptera crown group appeared in the Late Carboniferous (∼300 Ma) and fed on nonvas-cular land plants. Lepidoptera evolved the tube-like proboscis in the Middle Triassic (∼241 Ma), which allowed them to acquire nectar from flowering plants. This morphological innovation, along with other traits, likely promoted the extraordinary diversification of superfamily-level lepidopteran crown groups. The ancestor of butterflies was likely nocturnal, and our results indicate that butterflies became day-flying in the Late Cretaceous (∼98 Ma). Moth hearing organs arose multiple times before the evolutionary arms race between moths and bats, perhaps initially detecting a wide range of sound frequencies before being co-opted to specifically detect bat sonar. Our study provides an essential framework for future comparative studies on butterfly and moth evolution. Lepidoptera | coevolution | phylogeny | angiosperms | bats
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Spilomelinae and Pyraustinae form a species-rich monophylum of Crambidae (snout moths). Morphological distinction of the two groups has been difficult in the past, and the morphologically heterogenous Spilomelinae has not been broadly accepted as a natural group due to the lack of convincing apomorphies. In order to investigate potential apomorphic characters for Spilomelinae and Pyraustinae and to examine alternative phylogenetic hypotheses, we conduct a phylogenetic analysis using 6 molecular markers and 114 morphological characters of the adults representing 77 genera of Spilomelinae and 18 genera of Pyraustinae. The results of the analysis of the combined data strongly suggest that Spilomelinae and Pyraustinae are each monophyletic and sister to each other. Wurthiinae is confirmed as ingroup of Spilomelinae, and Sufetula Walker, 1859 as a non-spilomeline. Within Spilomelinae, several well supported clades are obtained, for which we propose a first phylogeny-based tribal classification, using nine available and four new names: Hydririni Minet, 1982 stat.rev., Lineodini Amsel, 1956 stat.rev., Udeini trib.n., Wurthiini Roepke, 1916 stat.rev., Agroterini Acloque, 1897 stat.rev., Spilomelini Guenée, 1854 stat.rev. (= Siginae Hampson, 1918), Herpetogrammatini trib.n., Hymeniini Swinhoe, 1900 stat.rev., Asciodini trib.n., Trichaeini trib.n., Steniini Guenée, 1854 stat.rev., Nomophilini Kuznetzov & Stekolnikov, 1979 stat.rev. and Margaroniini Swinhoe & Cotes, 1889 stat.rev. (=Dichocrociinae Swinhoe, 1900; = Hapaliadae Swinhoe, 1890; = Margarodidae Guenée, 1854). The available name Syleptinae Swinhoe, 1900 could not be assigned to any of the recovered clades. Three tribes are recognized in Pyraustinae: Euclastini Popescu-Gorj & Constantinescu, 1977 stat.rev., Portentomorphini Amsel, 1956 stat.rev. and Pyraustini Meyrick, 1890 stat.rev. (= Botydes Blanchard, 1840; = Ennychites Duponchel, 1845). The taxonomic status of Tetridia Warren, 1890, found to be sister to all other investigated Pyraustinae, needs further investigation. The four Spilomelinae tribes that are sister to all other, ‘euspilomeline’ tribes share several plesiomorphies with Pyraustinae. We provide morphological synapomorphies and descriptions for Spilomelinae, Pyraustinae and the subgroups recognised therein. These characters allow the assignment of additional 125 genera to Spilomelinae tribes, and additional 56 genera to Pyraustinae tribes. New and revised combinations are proposed: Nonazochis Amsel, 1956 syn.n. of Conchylodes Guenée, 1854, with Conchylodes graph­ialis (Schaus, 1912) comb.n.; Conchylodes octonalis (Zeller, 1873) comb.n. (from Lygropia); Hyperectis Meyrick, 1904 syn.n. of Hydriris Meyrick, 1885, with Hydriris dioctias (Meyick, 1904) comb.n., and Hydriris apicalis (Hampson, 1912) comb.n.; Conogethes pandamalis (Walker, 1859) comb.n. (from Dichocrocis); Arthromastix pactolalis (Guenée,1854) comb.n. (from Syllepte); Prophantis coenostolalis (Hampson, 1899) comb.n. (from Thliptoceras); Prophantis xanthomeralis (Hampson, 1918) comb.n. (from Thliptoceras); Prophantis longicornalis (Mabille, 1900) comb.n. (from Syngamia); Charitoprepes apicipicta (Inoue, 1963) comb.n. (from Heterocnephes); Prenesta rubrocinctalis (Guenée, 1854) comb.n. (from Glyphodes); Alytana calligrammalis (Mabille, 1879) comb.n. (from Analyta). Epherema Snellen, 1892 stat.rev. with its type species E. abyssalis Snellen, 1892 comb.rev. is removed from synonymy with Syllepte Hübner, 1823. Ametrea Munroe, 1964 and Charitoprepes Warren, 1896 are transferred from Pyraustinae to Spilomelinae; Prooedema Hampson, 1891 from Spilomelinae to Pyraustinae; Aporocosmus Butler, 1886 from Spilomelinae to Odontiinae; Orthoraphis Hampson, 1896 from Spilomelinae to Lathrotelinae; Hydropionea Hampson, 1917, Plantegumia Amsel, 1956 and Munroe’s (1995) “undescribed genus ex Boeo­tarcha Meyrick” are transferred from Spilomelinae to Glaphyriinae.
Understanding the evolutionary mechanisms governing the uneven distribution of species richness across the tree of life is a great challenge in biology. Scientists have long argued that sexual conflict is a key driver of speciation. This hypothesis, however, has been highly debated in light of empirical evidence. Recent advances in the study of macroevolution make it possible to test this hypothesis with more data and increased accuracy. In the present study, we use phylogenomics combined with four different diversification rate analytical approaches to test whether sexual conflict is a driver of speciation in brush-footed butterflies of the tribe Acraeini. The presence of sphragis, an external mating plug found in most species among Acraeini, was used as a proxy for sexual conflict. Diversification analyses statistically reject the hypothesis that sexual conflict is associated with shifts in diversification rates in Acraeini. This result contrasts with earlier studies and suggests that the underlying mechanisms driving diversification are more complex than previously considered. In the case of butterflies, natural history traits acting in concert with abiotic factors possibly play a stronger role in triggering speciation than does sexual conflict.
The frenulum is a wing coupling structure that is found on the wings of most families of Lepidoptera. It is a single bristle or set of bristles that originate from the base of the hindwing that often interlocks with the forewing during flight. This wing coupling mechanism is thought to have been a major evolutionary innovation that allowed for enhanced flight in Lepidoptera. The sack‐bearer moths (Mimallonidae) are unusual among Lepidoptera in that not all species within the family have a frenulum. We test the hypothesis that the frenulum is not necessary and is therefore lost in mimallonids that have longer male forewings, because such wings are perhaps better suited to be coupled by other means. To understand the evolution of the frenulum, we inferred the most taxonomically and genetically sampled anchored hybrid enrichment‐based phylogeny of Mimallonidae, including 604 loci from all 41 genera and from 120 species, covering about 40% of the described species in the family. The maximum likelihood tree robustly supports major relationships within the family, and ancestral state reconstruction clearly recovers the frenulum as the plesiomorphic condition in Mimallonidae. Our results show that the frenulum is more often observed in species that have shorter, rather than longer, male forewings. The frenulum has historically been used as an important character for intrafamilial classification in Mimallonidae, but our results conclusively show that this character system is more variable than previously thought. Based on our results, we erect two new subfamilies, Roelofinae St Laurent & Kawahara, subfam.n. and Meneviinae St Laurent, Herbin, & Kawahara, subfam.n., for four genera previously considered incertae sedis. In the predominantly frenulum‐lacking clade Cicinninae, we describe a new genus, Cerradocinnus St Laurent, Mielke, & Kawahara, gen.n., and the genus Gonogramma stat. rev. is revalidated to include many species previously placed in Cicinnus sensu lato. With these changes, Cicinnus can now be considered monophyletic. Thirty‐three species are transferred to Gonogramma from Cicinnus sensu lato. This published work has been registered on Zoobank,‐DA6A‐4814‐A312‐36CBAA168B8B. We inferred the most taxonomically and genetically sampled anchored hybrid enrichment‐based phylogeny of Mimallonidae, including 604 loci from all 41 genera, covering about 40% of the species in the family. Ancestral state reconstructions of frenulum presence/absence and statistical tests revealed that the frenulum is more often observed in species that have shorter, rather than longer, male forewings. We describe two new subfamilies for genera previously considered incertae sedis, describe a new genus, and revalidate Gonogramma to include many species formerly placed in Cicinnus.
While the references in the following chapters of these volumes by necessity will be focussed on twentieth-century works, a brief outline is given below of principal pre-1900 contributions to the foundation of contemporary lepidopterology (as covered in the present Handbook). © 1998 by Walter de Gruyter GmbH & Co., Berlin. All right reserved.
Anania hortulata (Linnaeus, 1758) (Lepidoptera: Crambidae: Pyraustinae) is a strikingly coloured, common, and widespread species that has long been recognised as a single species widely distributed in Asia, Europe, and North America. Using a combination of molecular and morphometric data, this study resolved that A. hortulata is actually a species complex of two superficially indistinguishable species. Phylogenetic and network analyses based on the mitochondrial COI gene discriminated lineages from all major geographical regions of China as distinct, A. sinensis Yang and Landry new species , whereas A. hortulata occurs in Central Asia, Europe, and North America. Nuclear gene (CAD) and morphological differences in the genital characters provided further evidence for the separation of A. hortulata and A. sinensis .
Relationships within satyrine butterflies have been notoriously difficult to resolve using both morphology and Sanger sequencing methods, and this is particularly true for the mainly Neotropical subtribe Euptychiina, which contains about 400 described species. Known larvae of Euptychiina feed on grasses and sedges, with the exception of the genus Euptychia, which feed on mosses and lycopsids, and the butterflies occur widely in rainforest, cloudforest and grassland habitats, where they are often abundant. Several previous molecular and morphological studies have made significant progress in tackling the systematics of the group, but many relationships remain unresolved, with long-branch-attraction artifacts being a major problem. Additionally, the monophyly of the clade remains uncertain, with Euptychia possibly not being closely related to the remainder of the clade. Here we present a backbone phylogeny of the subtribe based on 106 taxa, 368 nuclear loci, and over 180,000 bps obtained through hybrid enrichment. Using both concatenation and species tree approaches (IQ-TREE, EXABAYES, ASTRAL), we can for the first time strongly confirm the monophyly of Euptychiina with Euptychia being the sister group to the remainder of the clade. The Euptychiina is divided into nine well supported clades, but the placement of a few genera such as Hermeuptychia, Pindis and the Chloreuptychia catharina group still remain uncertain. As partially indicated in previous studies, the genera Cissia, Chloreuptychia, Magneuptychia, Megisto, Splendeuptychia and Euptychoides, among others, were found to be highly polyphyletic and revisions are in preparation. The phylogeny will provide a strong backbone for the analysis of datasets in development that are much more taxonomically comprehensive but have orders of magnitude fewer loci. This study therefore represents a critical step towards resolving the higher classification and studying the evolution of this highly diverse lineage.
The origins and evolution of Hawaiian biodiversity are a matter of controversy, and the mechanisms of lineage diversification for many organisms on this remote archipelago remain unclear. Here we focus on the poorly known endemic leaf-mining moth genus Philodoria (Lepidoptera, Gracillariidae), whose species feed on a diversity of Hawaiian plant lineages, many of which are critically endangered. We use anchored hybrid enrichment to assemble the first phylogenomic dataset (507 loci) for any Hawaiian animal taxon. To uncover the timing and pattern of diversification of these moths, we apply two frequently used dating calibration strategies, biogeographic calibrations and secondary calibrations. Island calibrations on their own resulted in much younger and unrealistic dates compared to strategies that relied on secondary calibrations. Philodoria probably originated on the now partially sunken islands of Laysan or Lisianski, approximately 21 Ma, and were associated with host plants in the families Ebenaceae, Malvaceae or Primulaceae. Major feeding groups associated with specific host-plant families originated soon after the plants colonized the islands. Allopatric isolation and host shifts, in concert and independently, probably play major roles in the diversification of Philodoria Our dating results indicate that Philodoria is among the oldest known Hawaiian arthropod lineages, and that island calibrations alone can lead to unrealistically young dates.