Revision of taxonomic status of Anthrenus pimpinellae isabellinus (Coleoptera: Dermestidae)

Abstract

Abstract. For 160 years, Anthrenus pimpinellae isabellinus Küster, 1848 has been considered a subspecies of A. pimpinellae Fabricius, 1775. However, habitus shape differs between the subspecies with A. p. isabellinus being broader than A. p. pimpinellae
and resembling more closely A. dorsatus Mulsant & Rey, 1868. Here A. p. pimpinellae and A. p. isabellinus, are examined to look
for evidence that they comprise a single taxonomic unit. Habitus and genital structures are considered, and the universal animal
barcode region of the mitochondrial cytochrome oxidase I gene is sequenced. The results of the morphological, morphometric,
and genetic analyses mirror each other perfectly and suggest that A. p. isabellinus is the same species as A. dorsatus rather than
being a subspecies of A. pimpinellae. The very small intraspecifi c DNA sequence variation supports the view that A. dorsatus
and A. p. isabellinus belong to a single species that diverges considerably from A. p. pimpinellae. Morphology, including genital
structure, is congruent with the genetic data and provides a powerful way of resolving species organisation in these widespread
beetles. In view of these fi ndings, Anthrenus isabellinus Küster, 1848 is restored to full species status and Anthrenus dorsatus
Mulsant & Rey, 1868 becomes its new junior subjective synonym.

Full text

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EUROPEAN JOURNAL OF ENTOMOLOG
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contains many phenotypically similar species within the
A. pimpinellae species complex. All its species carry a
mixture of black, orange (yellow or brown) and white (or
off-white) scales, and nearly all species have most of the
white scales on the dorsal surface arranged as a broad band
across the elytra. Some of these species are suffi ciently
similar in appearance to have caused identifi cation issues
since A. pimpinellae Fabricius, 1775 was fi rst described
(Beal, 1998; Kadej et al., 2007).
Anthrenus pimpinellae is currently split into two well-
known subspecies based on coloration: A. p. pimpinellae
and A. p. isabellinus Küster, 1848. Küster (1848) origi-
nally described A. isabellinus as a separate species but it
was quickly relegated to a subspecies of A. pimpinellae
by Schaum (1862). It is not clear on what basis Schaum
Revision of taxonomic status of Anthrenus pimpinellae isabellinus
(Coleoptera: Dermestidae)
GRAHAM J. HOLLOWAY 1, DIMITRIOS E. BAKALOUDIS 2, MAXWELL V.L. BARCLAY 3, IVAN CAÑADA LUNA4,
CHRISTOPHER W. FOSTER 1, MARCIN KADEJ 5, AMANDA CALLAGHAN 1 and ROBERT J. PAXTON 6
1 Centre for Wildlife Assessment and Conservation, School of Biological Sciences, Harborne Building, Whiteknights, University
of Reading, Reading RG6 6AS, UK; e-mails: g.j.holloway@reading.ac.uk, c.w.foster@reading.ac.uk, a.callaghan@reading.ac.uk
2 Aristotle University of Thessaloniki, School of Forestry and Natural Environment, PO Box 241, University Campus,
541 24 Thessaloniki, Greece; e-mail: debakaloudis@for.auth.gr
3 Department of Life Sciences, Natural History Museum, London SW7 5BD, UK; e-mail: m.barclay@nhm.ac.uk
4 44 General Riera Street, Palma, 07003 Mallorca, Spain; e-mail: ivan_cl9@hotmail.com
5 Department of Invertebrate Biology, Evolution and Conservation, Faculty of Biological Science, University of Wrocław,
Przybyszewskiego 65, PL-51-148 Wrocław, Poland; e-mail: marcin.kadej@uwr.edu.pl
6 Institute for Biology, Martin Luther Universität Halle-Wittenberg, Hoher Weg 8, 06100 Halle (Saale), Germany;
e-mail: robert.paxton@zoologie.uni-halle.de
Key words. Coleoptera, Dermestidae, Anthrenus dorsatus, Anthrenus pimpinellae, Anthrenus isabellinus, taxonomy, new
synonymy, morphology, barcode, genitalia, antennae, sternite IX, species concept
Abstract. For 160 years, Anthrenus pimpinellae isabellinus Küster, 1848 has been considered a subspecies of A. pimpinellae Fa-
bricius, 1775. However, habitus shape differs between the subspecies with A. p. isabellinus being broader than A. p. pimpinellae
and resembling more closely A. dorsatus Mulsant & Rey, 1868. Here A. p. pimpinellae and A. p. isabellinus, are examined to look
for evidence that they comprise a single taxonomic unit. Habitus and genital structures are considered, and the universal animal
barcode region of the mitochondrial cytochrome oxidase I gene is sequenced. The results of the morphological, morphometric,
and genetic analyses mirror each other perfectly and suggest that A. p. isabellinus is the same species as A. dorsatus rather than
being a subspecies of A. pimpinellae. The very small intraspecifi c DNA sequence variation supports the view that A. dorsatus
and A. p. isabellinus belong to a single species that diverges considerably from A. p. pimpinellae. Morphology, including genital
structure, is congruent with the genetic data and provides a powerful way of resolving species organisation in these widespread
beetles. In view of these fi ndings, Anthrenus isabellinus Küster, 1848 is restored to full species status and Anthrenus dorsatus
Mulsant & Rey, 1868 becomes its new junior subjective synonym.
ZooBank Article Registration: http://zoobank.org/urn:lsid:zoobank.org:pub:4519E16A-7440-4646-BAB3-4FEF90949730
INTRODUCTION
The Dermestidae is a moderately large family of beetles
with the number of species currently claimed to lie be-
tween 1600 and 1700 (Háva, 2015, 2020), but the taxono-
my of parts of the family is poorly understood. Anthrenus
is a relatively large genus within the Dermestidae number-
ing about 260 species (Háva, 2020). Anthrenus provides
an example of complex, unresolved, taxonomy and is split
into 10 subgenera. Kadej (2018) carried out an examina-
tion of the genus Anthrenus using larval characteristics and
concluded that only the species within the subgenus An-
threnus were monophyletic, all other subgenera forming a
polyphyletic assemblage.
Even though the subgenus Anthrenus appears to be
monophyletic, it is not without its diffi culties. Notably, it
Eur. J. Entomol. 117: 481–489, 2020
doi: 10.14411/eje.2020.051
ORIGINAL ARTICLE

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Holloway et al., Eur. J. Entomol. 117 : 481–489, 2020 doi: 10.14411/eje.2020.051
gene) to establish phylogenetic relationships among the species.
The nomenclature and zoogeography follow Háva (2015), and
the conventional nomenclature, including of the taxa under study,
is used.
Study insects
Material was collected from around Thessaloniki (Greece),
Mallorca (Spain), and Maryland (the United States of America),
supplemented with preserved specimens from the Natural History
Museum (NHM), London. From the fi eld, specimens were almost
exclusively collected from white fl owers such as Hoary Cress
(Lepidium draba L., Brassicaceae) and Hemlock (C. maculatum)
(see Holloway & Bakaloudis, 2019; Holloway et al., in press).
Dermestidae were knocked from the fl owers into a plastic tray to
facilitate aspiration using a pooter. All fi eld collected specimens
along with those from the NHM were used for the morphological
analysis.
Only in Greece were A. p. pimpinellae, A. p. isabellinus and
A. dorsatus found together in the fi eld, along with Anthrenus
scrophulariae albidus Brullé, 1832. In the laboratory, the Greek
insects collected from Sindos, near Thessaloniki (40.673368N,
22.806583E) were separated by species/subspecies and retained
on a mixture of dead insects, feathers, and bone and blood meal.
When the adult insects died, they were removed from the breed-
ing medium and stored in 2% acetic acid until dissection. F1 off-
spring of each species/subspecies were reared through to produce
insects for genetic analysis. When insects emerged from their
pupal cases, they were fl ash frozen at –30°C then stored in 99%
ethanol to preserve the DNA. Prior to homogenisation to extract
the DNA, some specimens were quickly dissected (see morpho-
metric analysis) under a Brunel BMSL zoom stereo LED micro-
scope to confi rm species identity.
(1862) concluded that A. p. pimpinellae and A. p. isabel-
linus were conspecifi c. No examination of the genitalia ap-
pears to have been carried out, the colour patterns differ,
and the distributions overlap. Anthrenus p. pimpinellae is
claimed to have a cosmopolitan distribution (Háva, 2020),
whereas A. p. isabellinus is distributed around the western
Mediterranean (Háva, 2020). In other words, the subspe-
cies are sympatric in the western Mediterranean and, ac-
cording to theory (Mallet, 1995, 2008), should not be able
to retain integrity.
In previous studies, A. dorsatus Mulsant & Rey, 1868,
another species in the A. pimpinellae complex, has been
noted both in Greece (Holloway & Bakaloudis, 2019) and
in USA (Holloway et al., in press). In both locations, A.
p. isabellinus was found alongside A. dorsatus on fl owers
of Hemlock, Conium maculatum L. (Apiaceae). Both A. p.
isabellinus and A. dorsatus are broad-bodied, considerably
broader than A. p. pimpinellae (Holloway & Bakaloudis,
2020). Combined, this evidence suggests that the subspe-
cifi c association of A. p. pimpinellae and A. p. isabellinus
needs to be questioned. A study was carried out to establish
the true taxonomic relationship between A. p. pimpinellae,
A. p. isabellinus, and A. dorsatus.
MATERIALS AND METHODS
A three-pronged approach is taken: (1) to examine the genital
structure, supplemented by other features such as antennal struc-
ture, (2) to carry out a morphometric examination, and (3) to se-
quence a fragment of the mitochondrial COI gene (the universal
animal barcode region of the mitochondrial cytochrome oxidase I
Table 1. List of DNA sequenced Anthrenus specimens whose parents were collected at Sindos (Greece). “% ID to nominal species” is the
GenBank % DNA sequence identity to the colour-based species identity, except in A. dorsatus (values asterisked) whose % sequence ID
is to Anthrenus pimpinellae (the highest GenBank sequence ID to all Anthrenus dorsatus sequences) because GenBank does not contain
any Anthrenus dorsatus sequences. ND – sex not determined. Reference specimens (parents and additional F1 offspring from the same
breeding vial) for sequenced individuals are located in the private collection of G.J. Holloway.
Label on
Fig. 5
Identifi cation based
on colour pattern Sex
% ID to
nominal
species
Identifi cation based on
genitalia (Figs 1–3) and
morphometrics (Fig. 4)
Identifi cation based on
DNA barcode (in Fig. 5)
BOLD
sample ID
AN27 Anthrenus dorsatus ND 86.83* Anthrenus dorsatus Anthrenus dorsatus GJHAN27
AN32 Anthrenus dorsatus ND 86.25* Anthrenus dorsatus Anthrenus dorsatus GJHAN32
AN33 Anthrenus dorsatus ND 86.91* Anthrenus dorsatus Anthrenus dorsatus GJHAN33
AN34 Anthrenus dorsatus ND 86.59* Anthrenus dorsatus Anthrenus dorsatus GJHAN34
AN36 Anthrenus dorsatus ND 86.62* Anthrenus dorsatus Anthrenus dorsatus GJHAN36
AN9 Anthrenus pimpinellae isabellinus ♂86.30 Anthrenus dorsatus Anthrenus dorsatus GJHAN09
AN10 Anthrenus pimpinellae isabellinus ♀86.26 Anthrenus dorsatus Anthrenus dorsatus GJHAN10
AN11 Anthrenus pimpinellae isabellinus ♂86.28 Anthrenus dorsatus Anthrenus dorsatus GJHAN11
AN12 Anthrenus pimpinellae isabellinus ♂86.21 Anthrenus dorsatus Anthrenus dorsatus GJHAN12
AN22 Anthrenus pimpinellae isabellinus ♂86.43 Anthrenus dorsatus Anthrenus dorsatus GJHAN22
AN28 Anthrenus pimpinellae isabellinus ND 86.45 Anthrenus dorsatus Anthrenus dorsatus GJHAN28
AN29 Anthrenus pimpinellae isabellinus ND 86.47 Anthrenus dorsatus Anthrenus dorsatus GJHAN29
AN14 Anthrenus pimpinellae pimpinellae ♂99.84 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN14
AN15 Anthrenus pimpinellae pimpinellae ♀99.84 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN15
AN16 Anthrenus pimpinellae pimpinellae ♀100 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN16
AN18 Anthrenus pimpinellae pimpinellae ♀99.53 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN18
AN19 Anthrenus pimpinellae pimpinellae ♂99.84 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN19
AN20 Anthrenus pimpinellae pimpinellae ♂99.84 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN20
AN21 Anthrenus pimpinellae pimpinellae ♀99.84 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN21
AN23 Anthrenus pimpinellae pimpinellae ♂99.84 Anthrenus p. pimpinellae Anthrenus p. pimpinellae GJHAN23
AN24 Anthrenus scrophulariae albidus ND 98.83 n/a Anthrenus scrophulariae GJHAN24
AN25 Anthrenus scrophulariae albidus ND 99.00 n/a Anthrenus scrophulariae GJHAN25
AN26 Anthrenus scrophulariae albidus ND 99.17 n/a Anthrenus scrophulariae GJHAN26
AN37 Anthrenus scrophulariae albidus ND 98.89 n/a Anthrenus scrophulariae GJHAN37
AN38 Anthrenus scrophulariae albidus ND 98.09 n/a Anthrenus scrophulariae GJHAN38

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Morphological and morphometric analysis
The following specimens were used in the morphometric anal-
ysis: Anthrenus pimpinellae pimpinellae: Greece 9 males (fi eld
collected, Thessaloniki), France 1 male (NHM collection); An-
threnus pimpinellae isabellinus: Mallorca 2 males (fi eld collect-
ed, Pollensa), Greece 1 male (fi eld collected, Thessaloniki), US
1 male (fi eld collected, Maryland), Spain 1 male (NHM collec-
tion), 1 male labelled ‘Europe’ (NHM collection), Algeria 1 male
(NHM collection), Morocco 1 male (NHM collection); A. dor-
satus: Greece 9 males (fi eld collected, Thessaloniki), Mallorca 9
males (fi eld collected, Pollensa), Spain 1 male (NHM collection).
Identifi cation was based on genital and antennal structure,
along with habitus body plan and colouration. Dissection in-
volved detaching the abdomen from the rest of the insect using
two entomological micropins. The soft tergites were then peeled
off the harder ventrites to expose the aedeagus. Sternite IX was
also detached from the aedeagus. Images were taken using a
Canon EOS 1300D and stacked by Helicon Focus 6-Pro focus
stacking software. Habitus images were captured at ×20 magnifi -
cation and images of the antennae were recorded at ×63 magnifi -
cation. Images of the genitalia were captured at ×100 magnifi ca-
tion using a Brunel monocular SP28 microscope. Morphometric
measurements were taken using DsCap.Ink software. Identifi ca-
tion was confi rmed using Kadej et al. (2007), Holloway et al.
(2019) and Holloway & Bakaloudis (2019, 2020). All statistical
analyses were carried out using Minitab (version 19) software. To
increase stringency in statistical analyses between the same pair
of taxa, Bonferroni correction was applied to P values (Rosenthal
Fig. 1. Anthrenus pimpinellae ssp. pimpinellae, male, typical examples of (a) habitus coloration, (b) ventrites, (c) antenna, (d) aedeagus,
and (e) sternite IX. Scale bar = 1 mm for 1a and 1b, 100 μm for 1c, 1d, and 1e.

484
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& Rubin, 1987). Following correction, signifi cance levels p <
0.05, p < 0.01, and p < 0.001, are indicated as *, **, and ***,
respectively. Non-signifi cant results are indicated ns. Means and
standard errors are provided.
The following measurements were taken:
1. Body length (BL) (front edge of pronotum to tip of elytra)
2. Body width (BW) (maximum width across the elytra)
3. Paramere length (PL) (from the posterior tip of the para-
mere to the to the anterior end where the parameres meet)
Genetic analysis
DNA was extracted using a high salt protocol (Paxton et al.,
1996) from 25 adult beetles, bred in captivity from insects col-
lected from Sindos, near Thessaloniki (Greece), comprising A.
dorsatus (n = 5), A. p. isabellinus (n = 7), A. p. pimpinellae (n =
8) and A. scrophulariae albidus (n = 5; see Table 1). Sequenced
individuals comprised a mix of males and females that were de-
termined to species based on colour and morphology (Table 1).
Entire insects were crushed for DNA extraction; pinned reference
specimens relating to these samples are the parental generation
and other F1 offspring of the same breeding vial (G.J. Hollo-
way, private collection). The 25 samples were DNA barcoded at
COI, using standard protocols recommended by BOLD (http://
www.barcodinglife.org) with the animal barcode oligonucleotide
PCR primers LCO/HCO (Folmer et al., 1994). DNA sequences
were used to interrogate NCBI’s database using BLAST (https://
Fig. 2. Anthrenus dorsatus, male, typical examples of (a) habitus coloration, (b) ventrites, (c) antenna, (d) aedeagus, and (e) sternite IX.
Scale bar = 1 mm for 2a and 2b, 100 μm for 2c, 2d, and 2e.

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blast.ncbi.nlm.nih.gov/Blast.cgi) and the BOLD COI database
(http://www.barcodinglife.org). Beetles bred for sequencing were
uniquely numbered (Table 1) and therefore genetic analysis was
undertaken blind to species or subspecies identity.
Phylogenetic and evolutionary analyses of the 25 sequences
plus 7 additional reference sequences, including conspecifi cs [A.
(Anthrenus) festivus Erichson, 1846 from France, A. (A) pimpinel-
lae from Germany, A. (A) scrophulariae (Linnaeus, 1758) from
Germany, and two species of different subgenera (two sequences
each of Anthrenus (Nathrenus) verbasci (Linnaeus, 1767) from
Germany and Canada, and Anthrenus (Florilinus) museorum
(Linnaeus, 1761) both from Germany], retrieved from the NCBI
(GenBank) database, were conducted using MEGA version X
(Kumar et al., 2018; Stecher et al., 2020). Sequences were aligned
using ClustalW which, after removal of gaps, revealed a single
open reading frame in all sequences of 575 bases (191 amino
acids), suggesting sequence quality was good. Substitution model
selection by Maximum Likelihood showed the best model based
on AICc and BIC to be the TN93+G+I (Tamura-Nei model with
a discrete Gamma distribution to model evolutionary rate differ-
ences among sites, allowing for some sites to be evolutionarily
invariable), which was then employed to generate a phylogenetic
tree by Maximum Likelihood, with 500 bootstrap replicates to
estimate support for branches. The 25 new sequences generated
in this study are publicly available in BOLD (Table 1).
RESULTS
Figs 1, 2 and 3 show typical examples of A. p. pimpi-
nellae, A. dorsatus and A. p. isabellinus, respectively: (a)
habitus, (b) ventrites, (c) antenna, (d) aedeagus, and (e)
sternite IX.
Fig. 3. Anthrenus pimpinellae ssp. isabellinus, male, typical examples of (a) habitus coloration, (b) ventrites, (c) antenna, (d) aedeagus,
and (e) sternite IX. Scale bar = 1 mm for 3a and 3b, 100 μm for 3c, 3d, and 3e.

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The colour patterns of A. p. pimpinellae (Fig. 1a) and A.
dorsatus (Fig. 2a) consist of black, white and orange scales
in relatively similar distributions across the elytra. Dif-
ferences include the width of the trans-elytral white band
(narrower in A. p. pimpinellae than A. dorsatus), tightness
of packing of scales of the white band (spaced apart in A.
p. pimpinellae, overlapping in A. dorsatus), and the fi n-
ger of white scales joining the posterior edge of the white
band to the middle lateral elytral white spot (broken or ru-
dimentary in A. p. pimpinellae, complete in A. dorsatus).
The elytral colour pattern of A. p. isabellinus (Fig. 3a) is
distinctive, and much of the patterns shown in Figs 1a and
2a are covered in creamy coloured scales, tightly packed at
the base of the elytra and becoming more scattered towards
the elytral apices.
The ventrites of A. p. pimpinellae (Fig. 1b) are off-white
as a result of the mixing of white with pale brown scales.
At the lateral margins of the sternites are large patches of
black scales. The patch on sternite I is very large, meets the
lateral margin, and has no white scales on its anterior mar-
gin. The ventrites of A. p. isabellinus (Fig. 2b) and A. dor-
satus (Fig. 3b) are very similar in appearance; the bright
white scales are closely packed together (in A. p. pimpinel-
lae the scales are more spaced), the patches of black scales
along the lateral margins of each sternite are smaller than
those of A. p. pimpinellae, and the patches of black scales
on sternite I are sub-lateral, small and surrounded by white
scales.
The antennal clubs of A. dorsatus (Fig. 2c) and A. p. isa-
bellinus (Fig. 3c) are similar in shape and show wide su-
tures between the antennomeres (Kadej et al., 2007). The
antennal club of A. p. pimpinellae (Fig. 1c) is broader and
with narrow sutures between the antennomeres (Kadej et
al., 2007).
The aedeagus of A. p. pimpinellae (Fig. 1d) expands
from the base to the apex ending in broad, heavily hooked
parameres that are covered in shaggy hairs on the dorsal
surface. The aedeagi of A. dorsatus (Fig. 2d) and A. p.
pimpinellae (Fig. 3d) are very similar to each other. They
are narrower than A. p. pimpinellae, do not expand much
from the base to the apex, are not as broad and hooked as
A. p. pimpinellae, and have shorter, sparser hairs on the
dorsal surface.
The sternite IX of A. dorsatus (Fig. 2e) and A. p. isabel-
linus (Fig. 3e) are very similar to each other and show fl aps
between the anterior horns. Sternite IX of A. p. pimpinellae
(Fig. 1e) does not show these fl aps (Kadej et al., 2007).
Table 2 shows the morphometric values for A. p. pimpi-
nellae, A. p. isabellinus, and A. dorsatus. For all four of the
morphological metrics shown (BL, BW/BL, PL, PL/BL),
A. p. isabellinus deviates signifi cantly from A. p. pimpinel-
lae. Anthrenus p. isabellinus has a longer, rounder body,
longer parameres, and longer parameres relative to body
length than A. p. pimpinellae. For all four of these mor-
phological metrics, A. p. isabellinus does not differ signifi -
cantly from A. dorsatus.
Fig. 4 shows a plot of PL on BW/BL for all specimens
of all taxa together. There is an obvious split in the data,
with the values for A. p. pimpinellae occupying a different
area within the plot than A. dorsatus. The values for the A.
p. isabellinus specimens nestle comfortably within the A.
dorsatus data points and are clearly removed from those of
A. p. pimpinellae.
Genetic analysis
All 25 sequences gave high GenBank query coverage
(> 97%) and closest sequence identity (> 85%) to Anthre-
nus species (Table 1). The closest BLAST hits in GenBank
and the closest BOLD match of A. p. pimpinellae and A. s.
scrophulariae were to their respective species, consistently
with high sequence identity of > 98% (Table 1). GenBank
and BOLD databases lack reference barcode sequences for
A. dorsatus and A. p. isabellinus. Both A. dorsatus and A.
p. isabellinus had GenBank closest hits to A. pimpinellae,
but both with a relatively low sequence identity of < 87%
(Table 1). BOLD returned ‘no match’ for both taxa due
to lack of close identity to any reference sequence in the
BOLD database. Phylogenetic analysis of our CO1 bar-
code sequences (Fig. 5) revealed three clear clusters within
Anthrenus s. str.: A. p. pimpinellae, A. dorsatus plus A. p.
isabellinus, and A. s. scrophulariae, each with high boot-
strap support (100%). The average sequence variation
within the A. pimpinellae cluster was 0.07%; within the A.
dorsatus + A. p. isabellinus cluster it was 0.18%; and with-
Fig. 4. Paramere length (μm) against body width (BW) on body
length (BL) for male Anthrenus p. pimpinellae (black), Anthrenus
dorsatus (grey), and A. p. isabellinus (open).
Table 2. Morphometrics for Anthrenus p. pimpinellae, A. p. isabellinus, and A. dorsatus. The units for male body length (BL) are mm
(± SE), the units for paramere length (PL) are μm (± SE). BW – body width. Statistical comparisons (t tests plus df) of adjacent pairs of
values are provided. ns – not signifi cant, ** p < 0.01, *** p < 0.001.
pimpinellae isabellinus dorsatus
BL
BW/BL
PL
PL/BW
2.584 ± 0.008
0.682 ± 0.003
366.5 ± 7.9
0.143 ± 0.003
t16 = 3.98, **
t16 = 8.59, ***
t16 = 18.62, ***
t16 = 9.05, ***
3.000 ± 0.006
0.734 ± 0.006
542.3 ± 3.7
0.181 ± 0.003
t25 = 1.03, ns
t25 = 2.39, ns
t25 = 0.74, ns
t25 = 1.03, ns
3.101 ± 0.088
0.718 ± 0.003
537.4 ± 5.3
0.174 ± 0.003

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in the A. s. scrophulariae cluster it was 0.66%. The average
DNA sequence divergence between the A. p. pimpinellae
cluster and the A. dorsatus + A. p. isabellinus cluster was
13.35%. These values of high between-group sequence di-
vergence versus low within-group sequence variation cou-
pled to the well supported phylogeny are strong evidence
that the three clusters represent separate species and that A.
dorsatus and A. p. isabellinus are conspecifi c.
Distribution
Fig. 6 shows the known circum-Mediterranean distribu-
tions of A. p. isabellinus and A. dorsatus. There is exten-
sive overlap between A. dorsatus and A. p. isabellinus.
Taxonomy
As a result of the data set out above, we hereby restore
Anthrenus isabellinus Küster, 1848 to full species status
from a subspecies of A. pimpinellae and propose the fol-
lowing new synonymy: Anthrenus dorsatus Mulsant &
Rey, 1868 is a junior subjective synonym of Anthrenus isa-
bellinus Küster, 1848.
DISCUSSION
Here we examined two forms that have been accepted as
subspecies for nearly 160 years, but one subspecies (A. p.
isabellinus) was believed to exist wholly within the spatial
distribution of the nominotypical subspecies (A. p. pimpi-
nellae). We found that A. p. isabellinus specimens from a
variety of geographical locations bear little resemblance to
A. p. pimpinellae. They are, without question, different spe-
cies. Küster (1848) originally described A. isabellinus as a
full species and Beal (1998) speculated that A. p. pimpinel-
lae and A. p. isabellinus were different species. This study
has demonstrated that both Küster and Beal were correct.
We examined the specimens using three techniques:
morphological comparison, morphometrics, and genetic
analysis. Gratifyingly, all three approaches arrived at the
same conclusion, that A. p. isabellinus is not closely related
to A. pimpinellae and resembles A. dorsatus in all respects.
This fi nding is signifi cant since not all workers are able
to make genetic comparisons, but morphological and mor-
phometric comparisons are more widely available. Geni-
tal structure is one of the most important characters used
by taxonomists to differentiate among species (Arnqvist,
1998), and we demonstrate here that careful examination
of the genitalia is important in Anthrenus species identifi -
cation. Moreover, morphological and morphometric com-
parisons can produce results as sound as those based on
DNA sequence data.
Beal (1998) suggested that we probably have a poor un-
derstanding of the distribution of the nominotypical A. p.
Fig. 5. Phylogenetic tree of Anthrenus spp. based on COI barcode
sequences of 32 Anthrenus species (575 positions in the fi nal
dataset) inferred by Maximum Likelihood and Tamura-Nei model
of base substitution (Tamura & Nei, 1993). The tree with the high-
est log likelihood is shown, and the percentage of trees (> 80%) in
which the associated taxa clustered together is shown next to the
branches (500 bootstraps). The tree is drawn to scale, with branch
lengths measured in number of substitutions per site (scale bar as
%) and is midpoint rooted.

488
Holloway et al., Eur. J. Entomol. 117 : 481–489, 2020 doi: 10.14411/eje.2020.051
pimpinellae due to the degree to which workers confused
different species from the A. pimpinellae complex. Anthre-
nus p. pimpinellae is claimed to be almost cosmopolitan.
Specimens held in the NHM would suggest that this claim
might not be true (G.J. Holloway, pers. obs.). Holloway &
Bakaloudis (2020) showed that there is little intra-specifi c
variation in colour pattern in A. p. pimpinellae. The cur-
rent study demonstrated that A. p. pimpinellae is narrow in
shape relative to A. isabellinus, with brownish versus clean
white ventrites, respectively. These features, body shape
and colour pattern, can be quite easily assessed under fi eld
conditions. It is hoped that fi eld workers will be able to
utilise the information provided here to help to understand
the true distribution of A. p. pimpinellae.
The majority of A. isabellinus both from the fi eld (a sam-
ple of over 500 insects from across Europe, GJH unpubl.
data) and others reared through in the laboratory have a
colour pattern resembling Fig. 2a (80% of individuals).
The remaining A. isabellinus possess more whitish elytral
scales (20% of individuals) along a continuous gradient
of increasing numbers of paler elytral scales to the palest
specimens as shown in Fig. 3a. Furthermore, insects with
the colour patterns shown in Fig. 2a produce some offspring
resembling Fig. 3a, whilst parental insect resembling Fig.
3a produce some offspring resembling Fig. 2a. There is no
evidence that the different colour patterns are the result
of genetic polymorphism. A more parsimonious explana-
tion for the colour pattern gradient is phenotypic plasticity.
Colour pattern plasticity is very common across a range of
insect taxa. Many Lepidoptera display wing scale colour
plasticity in response to developmental period (Holloway
et al., 1993; Kemp & Jones, 2001), with paler wing scales
produced by individuals with the shortest developmental
periods. In fact, colour pattern plasticity in response to de-
velopmental period is common across many insect groups,
including Coleoptera (Holloway et al., 1995; Michie et al.,
2010), Diptera (Marriott & Holloway, 1998; Gibert et al.,
2007), and Hemiptera (Sorokor et al., 2013; Sibilia et al.,
2018). In many cases it has been shown that colour pattern
plasticity in insects has adaptive signifi cance (Brakefi eld &
Reitsma, 1991; Ottenheim et al., 1999; Sibilia et al., 2018).
More work is required to establish why and how the varia-
tion in colour pattern in A. isabellinus is produced.
ACKNOWLEDGEMENTS. We are very grateful to the Coleoptera
curatorial team of the Natural History Museum, London, for pro-
viding access to the collections in their care. We are tremendously
grateful to two anonymous referees and the editor for making ex-
cellent and constructive comments for ways of improving on the
original submission.
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Received March 25, 2020; revised and accepted November 30, 2020
Published online December 16, 2020