DNA barcoding identifies the first North American records of the Eurasian moth, Eupithecia pusillata (Lepidoptera: Geometridae)

2010·4.8 MB·English

by  Jeremy R Dewaard

J. Entomol. Soc. Brit. Columbia 107,December2010 25
DNA
barcoding identifies the first
North American records of the Eurasian moth,
Eupitheciapusillata (Lepidoptera: Geometridae)
JEREMY R. deWAARD* ^ LELAND M. HUMBLE' ^
and B. CHRISTIAN SCHMIDT^
ABSTRACT
The first North American records ofthejuniper pug moth, Eupitheciapusillata (Denis
&
Schiffermiiller, 1775) (Lepidoptera: Geometridae), brought to our attention using
DNA barcoding, are presented. Documentation and collection localities suggest it was
introduced, established, and likely has persisted, at least in the Greater Vancouver area
ofBritish Columbia since the mid-1970s. We discuss the integration ofDNA barcoding
into routine biosurveillance and forest insect surveys to prevent such delay in recogni-
—
tion ofnon-indigenous species in this case, 34 years.
Key Words: Eupithecia pusillata, Eupithecia interruptofasciata, Eupithecia niphado-
philata, juniper pug moth, Juniperus, non-indigenous species, invasive species, DNA
barcoding
INTRODUCTION
DNA
barcoding ofbiological specimens and Budinoff 2005), agromyzid leafminers
has demonstrated repeatedly its utility as a (Scheffer et al. 2006), tephritid fruit flies
molecular diagnostic technique that merits (Armstrong and Ball 2005; Barr 2009),
integration into biosurveillance programs. siricidwasps (Wilson and Schiff2010), true
In contrast to other molecular tools com- bugs (Nadel et al. 2010), and numerous taxa
monly employed for species identification of moths (Ball and Armstrong 2006; Si-
of intercepted organisms, DNA barcoding monsen et al. 2008; Humble et al. 2009;
is a generic and standardized approach that deWaard et al. 2009; Gilligan and Epstein
meets international standards ofdata quality 2009; Armstrong 2010). Here we report the
and transparency (Floyd et al. 2010). Sev- first North American records ofthejuniper
eral studies have demonstrated the efficacy pug moth, Eupithecia pusillata (Denis &
DNA
of this technique for detecting non- Schiffermiiller, 1775) revealed by
indigenous species and determining native barcoding.
provenance, for example in leeches (Siddall
MATERIALS AND METHODS
While compiling a DNA barcode library (deWaard et al., submitted), the cytochrome
for the Geometridae of British Columbia c oxidase subunit I (COI) sequences de-
' University ofBritish Columbia, Department ofForest Sciences, Forestry Sciences Centre, Vancouver,
BC,CanadaV6T 1Z4
" Royal British Columbia Museum, Entomology, 675 Belleville Street, Victoria, BC, Canada V8W 9W2
(email:[email protected])
^ Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Bumside Road,
Victoria, BC,CanadaV8Z 1M5 (email: [email protected])
^ Canadian Food Inspection Agency, Canadian National Collection ofInsects, Arachnids and Nematodes,
K.W. Neatby Building, 960 Carling Avenue, Ottawa, ON, Canada KIA 0C6 (email:
[email protected])
26 J. Emomoi. Soc. Brit. Coiimbia 107. DtxiMBtR 2010
ri\ed from two EupitJiecia specimens were Natural Resources Canada. Canadian Eorest
found to be di\ergent from known nati\e Ser\ice. Pacitlc Eorestr\ Centre. X'ictoria.
Euprheciu. The two sequences were com- BC). were dissected to examine the genita-
pared to a reference barcode database of lia following the methods gi\en b\ Lafon-
Lepidoptera barcodes using the identifica- taine (2004). Images ofgenitalia were taken
tion engine (BOLD-ID) of the Barcode of using a Leica M205C microscope equipped
Life Data Systems (BOLD) (Ratnasingham with a Leica DFC4Q0 camera kit and Leica
and Hebert 200"). and tentati\el\ identit'ied L.AS Montage s\stem that assembles multi-
as Eupithccia pusilluhu a Eurasian species ple images in successue planes of focus
not known to occur in Noah America. The into a single image with a large depth of
reference barcode database for Geometridae field. The specimens were \eritled b\ com-
used b\- BOLD-ID is contmualK \ahdated parison of the structure of genitalia with
b\ speciahsts to ensure accurate identitlca- specimens held m the CNC (Canadian Na-
tions. and is particular!) well parameterized tional Collection of Insects. Arachnids and
due to a global campaign to barcode the Nematodes. Ottawa. ON), and tlgures of
nearK 23,000 species of the famil\ (see piisilliiia m Skou (I'^^Sb) and Mirono\'
http: www.lepbarcoding.org geometridae (2003). Related species in the E. fiiphado-
index.php). The nine sequences with identi- phildta D\ar. 1^04 group (Bolte U»0) were
cal and near-identical matches from Europe ruled out b\ genitalic comparison to speci-
were obtained from Axel Hausmann mens in the CNC. as were other Nonh
(Zoological State Collection. Munich. Ger- .American species.
many) and Marko Mutanen (L'ni\ersit\ of Historical data associated with the
Oulu. Oulu. Einland) and combined with specimens were compiled from specimen
related North American specimens [soisu labels and Eorest Insect and Disease Sur\e\'
Bolte U^'-HV). A neighbour-joining tree was (EIDS) records (\'an Sickle et al. 2001).
constructed on BOLD using the K.imura-2- The single specimen from PECA. collected
parameter distance method (Eig. 1 ). b\ EIDS. is uniqueU identified b>- a regis-
To pursue continuation of the identit\ tration number (e.g. 6-*^-001^-01 ) that
ofthe specimens, the two putatne E. pusil- links the specimen to a EIDS sampling
Una specimens obtained from the RBCM form, completed at the time of sample col-
(Royal British Columbia Museum. N'ictoria. lection, as well as a rearing record docu-
BC) and PECA (Anhropod reference col- menting the status of laborator\ rearings.
lection. Pacitlc Eorestr) Centre (PEC). These records are held on tile at PEC.
RESULTS
Specimens examined: - label data The host recorded was commonjuni-
I
(handwrinen information in italics. indi\id- per [Jiijiiperus coninmnis L.): Remarks &
ual lines separated b\ comma, multiple S\mptonts state '".Attacking se\eral orna-
labels separated b\ " ' ): mentals with moderate damage". The date
No. -6-9-0019-01. Date 19 vii. ¥A.[D.] recorded on the specimen label is the date
S.1976 c. juniper. Port. Coquirlani BC ofadult eclosion. While the Rearing Record
Ac. No. PFC. 200"-02"l. indicates a second adult eclosed on S.\ii.~6
The specimen was initial1\ identitled as and was subsequenth spread, the specinten
Eupirhecia imicolor (Hulst). The EIDS re- could not be found m the PECA reference
cords document that this specimen was one collection.
of two adults reared from tl\e lar\ae and 1 r - label data:
tl\e pupae (10 indi\iduals m total) col- BC. N. \ ancou\er. 5 AUG 1986. C.S.
lected b\ the B.C. Eorest Ser\ice on Mt. Guppy ROYAL BRITISH. COLUMBLA
Burke. Pon Coquitlam dTM 10 53 54b MUSEUM. ENTQ^^l-125'3
.
[4Q.3. -122."]. Ele\ation ^00 ft), on 15 May This specimen was identitled as
J. Entomol. Soc. Brit. Columbia 107,December2010 27
BCZSM Lep22832
DE
BCZSM Lep22831
MM00781
I
MIV101785| Fl
MM08340
I
Eupitheciapusillata
PFC-2007-0271
CA:BC
ENT991-012573
BCZSM Lep03633 DE
-\ BCZSM Lep21945 IT
MM01786
MM08143 Fl
PFC-2007-0259
Dun-08-073 CA: BC
CNCLEP00035444 E. niphadophilata
1
UASM59396 CA:AB
Dun-08-081
CGWC-1141
CGWC-1155 CA:BC E. interruptofasciata
I
CGWC-1202
0.01 CGWC-1205
Figure 1. Neighbour-joining tree ofEupithecia pusillata and two closely related species, E.
niphadophilata andE. interruptofasciata. Tree was reconstmcted with the barcode fragment of
the cytochrome oxidase I (COI) gene. Sequences shaded in grey are from two individuals col-
lected in Vancouver, Canada. Abbreviations: DE - Gennany, Fl - Finland, IT - Italy, CA -
Canada, BC-British Columbia, AB -Alberta.
Eupithecia sp. in the collection before ten- Description: A small moth with a wing-
mm
tative assignment to Eupithecia intricata span of 16-22 (Mironov 2003) (Figs.
taylorata Swett by JRD. 2a, 2e). Forewing narrow, mostly shades of
Diagnosis: Eupithecia pusillata is most light brown with black transverse lines and
similar to E. niphadophilata and particu- oblong discal spot. Hindwing pale grey-
larly E. interruptofasciata, but a number of brown with weakly marked transverse lines
Eupithecia species are superficially very and variable discal spot. Abdomen pale
similar and identification should be based grayish brown with narrow black lateral
on examination of genitalia. Compared to stripes. Male genitalia (Fig. 2d) composed
E. interruptofasciata, which is structurally of broad valva with small ventral process,
most similar, the male 8'"^ stemite apical heavily sclerotized sacculus, vesica with
prongs are narrower, more blunt and the three horn-like comuti, simple aedeagus
apical cleft is shallower; the base of the (Fig. 2c) and elongated 8"^ sternite with two
stemite is also narrower overall with a shal- narrow apical processes (Fig. 2b). Female
lower medial invagination. The basal half genitalia composed of elongate and scle-
ofthe male vesica is armed with one spine, rotized bursa copulatrix (Fig. 2h) with small
not two as in E. interruptofasciata. In the spines at base and larger spines at margin.
female genitalia, the large spines on the left Ovipositor is simple with long setae (Fig.
side ofthe ductus bursae do not extend be- 2f). Terminal segment ofpupal case is stout
yond the mid-point of the ductus, but ex- with prominent lateral lobes and cremaster
tend beyond the midpoint in both E. inter- bearing four pairs ofhook-like setae (Figs.
ruptofasciata andE. niphadophilata. 2h, 2i).
28 J.Entomol. Soc.Brit. Columbia 107,December2010
Figure 2. Morphology ofEupithecia pusillata. a) male, dorsal view, b) male, 8'*' stemite, c)
male, aedeagus, d) male, genital capsule e) female, dorsal view, f) female, ovipositor, g) fe-
male, bursa copulatrix, h) pupa, terminal segment, dorsal view, i) pupa, terminal segment, lat-
eral view. Scale bars: a, e = 5 mm; b-d, f-i = 0.5 mm. A colour version of this figure is
available from Dr. Lee Humble.
Distribution and Habitat: In its native E. pusillata is present in the Caucasus
European range, the nominate subspecies is Mountains (Mironov 2003). In Asia, its
widely distributed from southern Europe, range extends across Russia from Sahkalin
its range extends to the Mediterranean from through Siberia, the Altai and Caucasus
eastern Spain to mainland Greece and Ro- regions (Skou 1986). The subspecies E.
mania, then extends north and west across pusillata scoriata Staudinger, 1857 has
northern Ukraine into western Russia. With been recorded only from Iceland and south-
the exception of Corsica, it has not been western Greenland (Mironov 2003). Mi-
recorded from the islands ofthe Mediterra- ronov et al. (2008) recently described a
nean. To the north it is present in the British third subspecies, E. pusillata kashniirica
Isles, through central Europe, north to Mironov and Ratzel from the Himalayas. In
northern Scandinavia, and into western natural settings, E. pusillata can be found in
Russia across the southern Kola Peninsula heaths, forest edges, rocky cliffs, and simi-
(Skou 1986; Mironov 2003; Karsholt & van lar habitats where the primary host grows.
Nieukerken 2010). A disjunct population of In urban areas, it can be common in gar-
J.Entomol. Soc. Brit.Columbia 107,December2010 29
dens. It is known from sea level up to ap- generally regarded as monophagous
proximately 2,500 m elevation in the Sierra (Mironov 2003), although it has also been
Nevada (Spain) and the Alps (Switzerland) recorded feeding on Douglas-fir, Pseu-
(Weigt 1993; Mironov 2003). dotsuga menziesii (Mirb.) Franco
Life History and Notes: The following (Pinaceae) in France (Roques et al. 2006),
data are based on European populations, where this North American tree is culti-
and it is expected that flight times, voltin- vated. The host of the subspecies scoriata
ism and larval hosts will be similar in North and kashmirica is not known, but is pre-
America, should extant populations be dis- sumed to also be Jimiperus. Eupithecia
covered. Univoltine, with larval stage from pusillata overwinters in the egg stage and
late April to mid-June and adult flight pe- pupates in a loose web in the ground (Skou
riod from mid-July to late September (Skou 1986). It is attacked by a variety ofichneu-
1986; Mironov 2003). As its common name monid and braconid species listed in Mi-
implies, the primary host ofE. pusillata is ronov (2003). It is not known ifother native
common juniper, Jimipenis communis L. or ornamental species ofJunipenis are suit-
(Cupressaceae) (Skou 1986), of which it able hosts in British Columbia.
feeds on young needles and flowers. It is
DISCUSSION
Eupithecia Curtis is a large genus with spread beyond the point of introduction.
1529 described species and subspecies Secondly, the locality ofthe first collection
(Scoble 1999; Scoble & Hausmann 2007), (Mt. Burke), the number of individuals re-
and about 160 species in North America corded (ten), and the damage observations
(Powell and Opler 2009). TheNorth Ameri- in the FIDS record, all indicate that there
can species were revised by McDunnough was an established E. pusillata population
(1949), and the Canadian fauna was revised in BC in 1976 (but note this is the only
by Bolte (1990). Eupitheciapusillata is part FIDS record of a Eupithecia on juniper
ofthe niphadophilata species group, which from greater Vancouver). And lastly, the
includes two Nearctic and one Palearctic 1986 collection from North Vancouver sug-
species (Bolte 1990), all feeding primarily gests that the population has persisted, or it
onjunipers (Skou 1986; Bolte 1990). did so for at least a decade. Subsequent
Although we currently have only two surveys, initially in the Vancouver area, are
specimens of Eupithecia pusillata from required to determine the contemporary
North America, we can extract a great deal status ofthis species.
of information from the associated data The excellent documentation of FIDS
documentation. First of all, the collections that enabled inferences about the status of
were made in urbanized Vancouver, BC, E. pusillata is unfortunately a relict of the
suggesting the species was introduced. The past; the program ceased in 1996 after al-
lack ofrecords, particularly from inland BC most 50 years ofoperation due to budgetary
(which is well-surveyed for macro- cut-backs (Van Sickle et al. 2001). Pro-
Lepidoptera), the Yukon Territory and grams such as this, based on surveying or
Alaska, lead us to conclude that the species inventorying diversity, are simultaneously
is not naturally Holarctic like some a) a tremendous resource for managers,
Eupithecia (see Skou 1986, Bolte 1990). foresters and scientists, and b) reliant on
Furthermore, the six Eupithecia species tremendous resources themselves particu-
considered Holarctic all show at least 1% larly in terms ofhighly qualified personnel
COI sequence divergence (data not shown) (e.g. Marshall et al. 1994). The present case
indicative of separation in the Pleistocene. illustrates the value of these long-term,
The absence ofadditional records also sug- well-documented biological surveys, but
gests that there has not been substantial these programs are often hindered by the
30 J. Entomol. Soc. Brit. Columbia 107, Df.cember2010
necessity to rear immatures to allow the are certain to make species diagnosis in this
DNA
diagnosis of species. Just as barcod- form time- and cost-effective. Furthermore,
ing makes an invaluable tool for biosurveil- most years of the FIDS program predated
lance (Floyd et al. 2010), it could likewise electronic databases, so it would also be
assist any regional or national biomonitor- better served by modem and online rela-
BOLD
ing program ofsimilar scope to FIDS. Bar- tional databases such as
coding could not only identify immature (Ratnasingham and Hebert 2007). With
stages (Ahrens et al. 2007) making rearing DNA barcoding in place, a resource similar
nonobligatory, it could also identify the to FIDS could once again be realized, and
plant meal of gut contents (Miller et al. without having to expend substantial re-
2007), identify parasitoids (Rougerie et al. sources as a cost. It would also, without
in press), and trace complex food webs question, speed th—e time ofnon-indigenous
(Sheppard et al. 2004; Smith et al. in press). species detection from years (34 in the
Decreasing costs and increasing capabilities case ofE. pusillata) to days.
of sequencing (e.g. Shokralla et al. 2010)
ACKNOWLEDGEMENTS
We are indebted to Axel Hausmann records. Rene Alfaro, Isabel Leal and two
(Zoological State Collection Munich) and anonymous reviewers provided valuable
Marko Mutanen (University of Oulu) for comments on the manuscript. Funding was
allowing us access to the Eurasian se- provided by a Forest Investment Account -
quences. We also wish to thank Stephanie Forest Science Program Student Grant and
Kirk for the specimen imaging, the staff at NSERC Graduate Scholarship (to JRD).
DNA
the Canadian Centre for Barcoding Molecular analyses were supported by the
for their assistance with genetic analysis, Canadian Barcode of Life Network from
and Sava Barudzija (Natural Resources Genome Canada through the Ontario Ge-
Canada, Canadian Forest Service, Frederic- nomics Institute, NSERC (to LMH), and
ton, NB) for extraction of historical FIDS other sponsors listed at www.BOLNET.ca.
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