Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, Ichneumonidae, Ephialtini) matched using DNA barcodes

Abstract

AbstractThe study of parasitoids and their hosts suffers from a lack of reliable taxonomic data. We use a combination of morphological characters and DNA sequences to produce taxonomic determinations that can be verified with reference to specimens in an accessible collection and DNA barcode sequences posted to the Barcode of Life database (BOLD). We demonstrate that DNA can be successfully extracted from consumed host spiders and the shed pupal case of a wasp using non-destructive methods. We found Acrodactyla
quadrisculpta to be a parasitoid of Tetragnatha
montana; Zatypota
percontatoria and Zatypota
bohemani both are parasitoids of Neottiura
bimaculata. Zatypota
anomala is a parasitoid of an as yet unidentified host in the family Dictynidae, but the host species may be possible to identify in the future as the library of reference sequences on BOLD continues to grow. The study of parasitoids and their hosts traditionally requires specialized knowledge and techniques, and accumulating data is a slow process. DNA barcoding could allow more professional and amateur naturalists to contribute data to this field of study. A publication venue dedicated to aggregating datasets of all sizes online is well suited to this model of distributed science.

Full text

Biodiversity Data Journal 1: e992
doi: 10.3897/BDJ.1.e992
Taxonomic paper
Spider hosts (Arachnida, Araneae) and wasp
parasitoids (Insecta, Hymenoptera,
Ichneumonidae, Ephialtini) matched using DNA
barcodes
Jeremy A. Miller , J. Dick M. Belgers , Kevin K. Beentjes , Kees Zwakhals , Peter van Helsdingen
† Naturalis Biodiversity Center, Leiden, Netherlands
‡ Wageningen University, Wageningen, Netherlands
§ Dr. Dreeslaan 204, Arkel, Netherlands
| European Invertebrate Survey, Leiden, Netherlands
Corresponding author: Jeremy A. Miller (jeremy.miller@naturalis.nl)
Academic editor: Lyubomir Penev
Received: 01 Sep 2013 | Accepted: 11 Sep 2013 | Published: 16 Sep 2013
Citation: Miller J, Belgers J, Beentjes K, Zwakhals K, van Helsdingen P (2013) Spider hosts (Arachnida,
Araneae) and wasp parasitoids (Insecta, Hymenoptera, Ichneumonidae, Ephialtini) matched using DNA
barcodes. Biodiversity Data Journal 1: e992. doi: 10.3897/BDJ.1.e992
Abstract
The study of parasitoids and their hosts suffers from a lack of reliable taxonomic data. We
use a combination of morphological characters and DNA sequences to produce taxonomic
determinations that can be verified with reference to specimens in an accessible collection
and DNA barcode sequences posted to the Barcode of Life database (BOLD). We
demonstrate that DNA can be successfully extracted from consumed host spiders and the
shed pupal case of a wasp using non-destructive methods. We found Acrodactyla
quadrisculpta to be a parasitoid of Tetragnatha montana; Zatypota percontatoria and Z.
bohemani both are parasitoids of Neottiura bimaculata. Zatypota anomala is a parasitoid of
an as yet unidentified host in the family Dictynidae, but the host species may be possible to
identify in the future as the library of reference sequences on BOLD continues to grow. The
study of parasitoids and their hosts traditionally requires specialized knowledge and
techniques, and accumulating data is a slow process. DNA barcoding could allow more
professional and amateur naturalists to contribute data to this field of study. A publication
† ‡ † § |
© Miller J et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

venue dedicated to aggregating datasets of all sizes online is well suited to this model of
distributed science.
Keywords
DNA barcode, host, morphological identification, non-destructive extraction, parasitoid
Introduction
Parasitoid wasps are among the most significant enemies of spiders (Foelix 2011). Among
the ichneumonid wasps, there are parasitoids of spider eggs, as well as ectoparasitoids of
post-embryonic spiders. The "Polysphincta group" of the tribe Ephialtini (also referred to by
its junior synonym Polysphinctini) informally refers to those ichneumonids that attack post-
embryonic spiders, typically of web-building species. These wasps develop as larvae
attached to the abdomen of the spider (Figs 1, 5, 9). This host-parasitoid relationship is
distinctive because the mobile spider continues to grow and develop along with the larval
parasitoid attached to its abdomen (koinobiont ectoparasitoid). Ultimately, the host is killed
and consumed by the larva just prior to pupation. In some cases, the behavior of the spider
is modified towards the end of its life to the advantage of the parasitoid (Eberhard 2000).
Figure 1.
Live Tetragnatha montana (RMNH.ARA.14127) parasitized by Acrodactyla quadrisculpta larva
(RMNH.INS.593867).
2 Miller J et al.

Figure 2.
Tetragnatha montana (RMNH.ARA.14127) preserved in alcohol after being consumed by
Acrodactyla quadrisculpta larva (RMNH.INS.593867). Overall view.
Figure 3.
Tetragnatha montana (RMNH.ARA.14127) preserved in alcohol after being consumed by
Acrodactyla quadrisculpta larva (RMNH.INS.593867). Detail of prosoma.
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 3

Figure 4.
Live adult female Acrodactyla quadrisculpta (RMNH.INS.593867).
Figure 5.
Live Tetragnatha montana (RMNH.ARA.14128) parasitized by Acrodactyla quadrisculpta larva
(RMNH.INS.593868).
4 Miller J et al.

Figure 6.
Tetragnatha montana (RMNH.ARA.14128) preserved in alcohol after being consumed by
Acrodactyla quadrisculpta larva (RMNH.INS.593868).
Figure 7.
Larval exuvium of Acrodactyla quadrisculpta (RMNH.INS.593868). DNA barcode sequence was
obtained from this specimen.
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 5

Most primary data associating spiders and their parasitoid species comes from rearing, i.e.,
keeping the host spider alive in the lab long enough for the wasp to mature (e.g., Bristow
1941, Nielsen 1923). This approach requires care, hard work, and expertise, and the
accumulation of data is a slow process (Fitton et al. 1987). Furthermore, in our experience
with "Polysphincta group" wasps, few spider hosts reach maturity before their parasitoids
do. This is unfortunate from the perspective of the biologist because most of the best
diagnostic morphological characteristics for spider species identification appear only in the
adult stage. As a result, positive identification of the host is often elusive and the literature
is blemished by dubious host-parasitoid association records (e.g., Shaw 2006).
Figure 8.
Live adult male Acrodactyla quadrisculpta (RMNH.INS.593868).
Figure 9.
Live Tetragnatha montana (RMNH.ARA.14129) parasitized by Acrodactyla quadrisculpta larva
(KZPC).
6 Miller J et al.

The advent of DNA barcoding offers a path to species determination where traditional
morphology falls short. Increasingly, studies of parasitoids and their hosts are turning to
DNA-based methods as an alternative or supplement to rearing (Santos et al. 2011, Hrcek
et al. 2011, Rougerie et al. 2011, Quicke et al. 2012). Here we apply DNA barcoding
techniques to positively associate parasitoids and their hosts. None of the spider hosts in
this study reached maturity before being killed by their parasitoid. We successfully
obtained DNA barcode sequences from the spider host remains left by the wasp just before
pupation (Figs 2, 3, 6, 10, 11, 14, 17). Adult wasps were identified using traditional
morphology.
Figure 10.
Tetragnatha montana (RMNH.ARA.14129) preserved in alcohol after being consumed by
Acrodactyla quadrisculpta larva.
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 7

Figure 11.
Neottiura bimaculata (RMNH.ARA.14036) preserved in alcohol after being consumed by Zatypota
percontatoria larva (RMNH.INS.593327).
Figure 12.
Silk coccoon of Zatypota percontatoria (RMNH.INS.593327), preserved specimen.
8 Miller J et al.

Figure 13.
Adult Zatypota percontatoria (RMNH.INS.593327), specimen.
Figure 14.
Neottiura bimaculata (RMNH.ARA.14037) preserved in alcohol after being consumed by Zatypota
bohemani larva (RMNH.INS.593328).
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 9

Figure 15.
Adult female Zatypota bohemani (RMNH.INS.593328), specimen.
Figure 16.
Adult female Zatypota bohemani (RMNH.INS.593328), specimen, detail of pronotum.
10 Miller J et al.

Materials and methods
In most cases, juvenile spiders observed to be hosting wasp larvae were collected live in
the field and taken to the lab for rearing (Figs 1, 5, 9). The adult wasp was allowed to fully
develop, killing the host spider in the process. In one record from France, the wasp had
already killed its host and pupated inside a loose silk cocoon (Figs 18, 19). In all cases, the
host spider died as a subadult and the body was left in poor condition, rendering positive
identification by means of traditional morphology unreliable (Figs 2, 3, 6, 10, 11, 14, 17).
DNA extractions were performed using the Thermo Labsystems KingFisher extraction
robot at the Naturalis Biodiversity Center DNA barcoding facility. For host spiders, DNA
was extracted by placing the entire specimen directly (without grinding) in lysis buffer with
proteinase K for the three hour incubation step. After incubation, the specimen was
returned to ethanol and the extraction continued using the lysis buffer solution. This caused
negligible further damage to the specimen. In one case, DNA was also extracted from a
wasp larval exuvium by this method (Fig. 7). For adult wasps, one leg was removed as
source tissue for DNA extraction. To obtain the standard animal DNA barcode fragment of
the mitochondrial cytochrome oxidase I gene (Hebert et al. 2003), PCR was performed
using either the primers LCO1490 (5'-GGTCAACAAATCATCATAAAGATATTGG-3')
(Folmer et al. 1994) and Chelicerate Reverse 2 (5'-
GGATGGCCAAAAAATCAAAATAAATG-3') (Barrett and Hebert 2005) (for spiders and
larval exuvium) or a coctail of primers LCO1490 (Hebert et al. 2003) and LepF (5'-
ATTCAACCAATCATAAAGATATTGG-3') paired with HCO2198 (5'-
TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994) and LepR1 (5'-
TGATTTTTTGGACATCCAGAAGTTTA-3') (Hebert et al. 2003) (for adult wasps). PCR
Figure 17.
Dictynidae sp. (RMNH.ARA.14254) preserved in alcohol after being consumed by Zatypota
anomala larva (RMNH.INS.593866). Arrow indicates calamistrum, a morphological structure found
in some dictynid species, but relatively few other European spiders.
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 11

reactions contained 18.75µl mQ, 2.5µ 10x PCR buffer CL, 1.0µl 25mM of each primer,
0.5µl 2.5mM dNTPs and 0.25µl 5U Qiagen Taq. PCR was performed using initial
denaturation of 180s at 94°C, followed by 40 cycles of 15s at 94°C, 30s at 50°C and 40s at
72°C, finished with a final extension of 300s at 72°C and pause at 12°C. Sequencing was
performed by Macrogen (http://www.macrogen.com
). For all barcoded specimens,
sequences, images, and collection data were uploaded to the Parasitoid Wasps and Spider
Hosts project (PWSH) on the Barcode of Life Database (BOLD; http://
www.boldsystems.org/). The "Species Level Barcode Records" search of the BOLD
database assisted in host spider identification. Where that was unsuccessful, we resorted
to the "All Barcode Records on BOLD" search option and Blast search on NCBI's Genbank
(http://www.ncbi.nlm.nih.gov/sites/gquery
). Historical literature records of host-parasitoid
associations relied on the World Ichneumonoidea database (Yu et al. 2012); primary
literature was typically not consulted. For Dutch specimens, latitude and longitude
coordinates are converted from the local RD (Rijksdriehoeksmeting) coordinate system; for
the French record, coordinates are latitude and longitude. All voucher specimens are
deposited in the collection of the Naturalis Biodiversity Center with the exception of one
wasp retained in the personal collection of Kees Zwakhals (KZPC).
Figure 18.
Zatypota anomala (RMNH.INS.593866) undergoing metamorphosis in the field.
12 Miller J et al.

Taxon treatments
Acrodactyla quadrisculpta (Gravenhorst, 1820)
•
Barcode of Life http://www.boldsystems.org/index.php/TaxBrowser_TaxonPage?
subtaxa=hidden&taxid=150402
•
Hymenoptera Online http://hol.osu.edu/?id=50298
Materials
a.
genus: Acrodactyla; specificEpithet: quadrisculpta; scientificNameAuthorship:
(Gravenhorst, 1820); country: Netherlands; stateProvince: Gelderland; locality:
Wageningen, Blauwe Kamer; decimalLatitude: 51.943995; decimalLongitude: 5.61874;
coordinateUncertaintyInMeters: 30; samplingProtocol: found (by beating) on fijnspar
(No
rway Spruce, Picea abies); eventDate: 04/17/2012; individualCount: 1; sex: female;
catalogNumber: RMNH.INS.593867; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.ARA.14127; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482005; institutionCode: RMNH; basisOfRecord: specimen
b.
genus: Acrodactyla; specificEpithet: quadrisculpta; scientificNameAuthorship:
(Gravenhorst, 1820); country: Netherlands; stateProvince: Gelderland; locality:
Wageningen, Blauwe Kamer; decimalLatitude: 51.943995; decimalLongitude: 5.61874;
coordinateUncertaintyInMeters: 30; samplingProtocol: found (by beating) on fijnspar
(No
rway Spruce, Picea abies); eventDate: 04/19/2012; individualCount: 1; sex: male;
catalogNumber: RMNH.INS.593868; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.ARA.14128; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482006; institutionCode: RMNH; basisOfRecord: specimen
c.
genus: Acrodactyla; specificEpithet: quadrisculpta; scientificNameAuthorship:
(Gravenhorst, 1820); country: Netherlands; stateProvince: Gelderland; locality:
Figure 19.
Silk coccoon of Zatypota anomala (RMNH.INS.593866) in the field.
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 13

Wageningen, Blauwe Kamer; decimalLatitude: 51.943995; decimalLongitude: 5.61874;
coordinateUncertaintyInMeters: 30; samplingProtocol: found (by beating) on fijnspar
(No
rway Spruce, Picea abies); eventDate: 04/19/2012; individualCount: 1; sex: male;
recordedBy: J. Dick M. Belgers; associatedOccurrences: RMNH.ARA.14129;
institutionCode: KZPC; basisOfRecord: specimen
Notes
Shaw (2006) notes the possibility of some confusion in the literature concerning this
parasitoid and the morphologically similar Acrodactyla carinator (Aubert, 1965). Errors
in identification of the parasitoid and/or host may be obscuring the true host specificity
of these wasps in some parts of their distribution.
Adult wasps were identified as A. quadrisculpta ( Figs 4, 8) by an experienced
ichneumonid taxonomist based on morphological characteristics with reference to the
taxonomic literature. DNA barcodes derived from two adult specimens
(RMNH.INS.593867 and RMNH.INS.593868) plus the larval exuvium (Fig. 7) were
used to query the BOLD database; no match was found. The barcode sequence from
the larval exuvium was identical to that of the adult (RMNH.INS.593868) except that
the last 9 bases on the 3' end were not sequenced. A subsequent Blast search of
NCBI's GenBank found sequences identified as A. quadrisculptata among the closest
matches. However, the closest matching sequences scored only 93% similarity, lower
than expected for most conspecific DNA barcodes. Of the 12 sequences with this
similarity score, three were identified as A. quadrisculpta; the remaining sequences
were less precisely identified but the taxonomic information was not in conflict with A.
quadrisculpta. Most if not all of the closest matching DNA barcode sequences were
derived from specimens collected in Manitoba, Canada. The resolution of these facts
might lie in a high genetic diversity within this widespread species, or taxonomic error
at some level. One specimen of A. quadrisculpta was not sequenced and remains in
the personal collection of Kees Zwakhals (KZPC).
Tetragnatha montana Simon, 1874
•
Barcode of Life http://www.boldsystems.org/index.php/TaxBrowser_TaxonPage?
subtaxa=hidden&taxid=178345
•
Araneae: Spiders of Europe http://www.araneae.unibe.ch/data/846/
Tetragnatha_montana
Materials
a.
genus: Tetragnatha; specificEpithet: montana; scientificNameAuthorship: Simon, 1874;
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.943995; decimalLongitude: 5.61874; coordinateUncertaintyInMeters:
30; samplingProtocol: found (by beating) on fijnspar (Norway Spruce, Picea abies);
eventDate: 03/30/2012; individualCount: 1; sex: female; lifeStage: juvenile;
catalogNumber: RMNH.ARA.14127; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.INS.593867; associatedSequences: http://
14 Miller J et al.

www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=2762428; institutionCode: RMNH; basisOfRecord: specimen
b.
genus: Tetragnatha; specificEpithet: montana; scientificNameAuthorship: Simon, 1874;
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.943995; decimalLongitude: 5.61874; coordinateUncertaintyInMeters:
30; samplingProtocol: found (by beating) on fijnspar (Norway Spruce, Picea abies);
eventDate: 04/01/2012; individualCount: 1; sex: female; lifeStage: juvenile;
catalogNumber: RMNH.ARA.14128; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.INS.593868; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=2762439; institutionCode: RMNH; basisOfRecord: specimen
c.
genus: Tetragnatha; specificEpithet: montana; scientificNameAuthorship: Simon, 1874;
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.943995; decimalLongitude: 5.61874; coordinateUncertaintyInMeters:
30; samplingProtocol: found (by beating) on fijnspar (Norway Spruce, Picea abies);
eventDate: 04/01/2012; individualCount: 1; sex: female; lifeStage: juvenile;
catalogNumber: RMNH.ARA.14129; recordedBy: J. Dick M. Belgers;
associatedSequences: http://www.boldsystems.org/index.php/
MAS_DataRetrieval_OpenSequence?selectedrecordid=2762449; institutionCode: RMNH;
basisOfRecord: specimen
Notes
A search of the BOLD database indicated that the host for all three Acrodactyla
quadrisculpta specimens was Tetragnatha montana Simon, 1874. Host sequences
scored 98.3%-100% similarity with 21 other data points identified as T. montana with
DNA barcode sequences available in BOLD (all private or early-release at the time of
writing). Some of the 100% matches were T. montana specimens sequenced as part of
a DNA barcoding study on Dutch spiders (Miller et al. 2013). Acrodactyla quadrisculpta
has been associated historically with a number of host species in the genus
Tetragnatha including T. montana (Yu et al. 2012).
Zatypota percontatoria (Müller, 1776)
•
Barcode of Life http://www.boldsystems.org/index.php/TaxBrowser_TaxonPage?
subtaxa=hidden&taxid=449209
•
Hymenoptera Online http://hol.osu.edu/index.html?id=50450
Material
a.
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.9428; decimalLongitude: 5.631533; coordinateUncertaintyInMeters:
30; eventDate: 2012-09-01; individualCount: 1; sex: female; lifeStage: adult;
catalogNumber: RMNH.INS.593327; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.ARA.14036; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482008; institutionCode: RMNH; basisOfRecord: specimen
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 15

Zatypota bohemani (Holmgren, 1860)
•
Barcode of Life http://www.boldsystems.org/index.php/TaxBrowser_TaxonPage?
subtaxa=hidden&taxid=469733
•
Hymenoptera Online http://hol.osu.edu/index.html?id=50427
Material
a.
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.94372; decimalLongitude: 5.619903; coordinateUncertaintyInMeters:
30; eventDate: 2012-07-05; individualCount: 1; sex: female; lifeStage: adult;
catalogNumber: RMNH.INS.593328; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.ARA.14037; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482010; institutionCode: RMNH; basisOfRecord: specimen
Neottiura bimaculata (Linnaeus, 1767)
•
Barcode of Life http://www.boldsystems.org/index.php/TaxBrowser_TaxonPage?
subtaxa=hidden&taxid=29775
•
Araneae: Spiders of Europe http://www.araneae.unibe.ch/data/56/Neottiura_bimaculata
Materials
a.
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.9428; decimalLongitude: 5.631533; coordinateUncertaintyInMeters:
30; eventDate: 2012-08-12; individualCount: 1; sex: female; lifeStage: juvenile;
catalogNumber: RMNH.ARA.14036; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.INS.593327; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482009; institutionCode: RMNH; basisOfRecord: specimen
b.
country: Netherlands; stateProvince: Gelderland; locality: Wageningen, Blauwe Kamer;
decimalLatitude: 51.94372; decimalLongitude: 5.619903; coordinateUncertaintyInMeters:
30; eventDate: 2012-06-14; individualCount: 1; sex: female; lifeStage: juvenile;
catalogNumber: RMNH.ARA.14037; recordedBy: J. Dick M. Belgers;
associatedOccurrences: RMNH.INS.593328; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482011; institutionCode: RMNH; basisOfRecord: specimen
Notes
A search of the BOLD database indicated that these two hosts are Neottiura
bimaculata (Linnaeus, 1767) ( Figs 11, 14). Host sequences scored 96.8%-99.5%
similarity with 20 other data points identified as N. bimaculata with DNA barcode
sequences available in BOLD including three specimens from a study on Dutch spiders
(Miller et al. 2013). Two parasitoid species were found to be associated with Neottiura
bimaculata: Zatypota percontatoria (Figs 12, 13) and Zatypota bohemani (Figs 15, 16).
16 Miller J et al.

Neither parasitoid had been associated previously with Neottiura bimaculata hosts (Yu
et al. 2012).
Zatypota anomala (Gravenhorst, 1820)
•
Barcode of Life (as Sinarachna anomala) http://www.boldsystems.org/index.php/
Taxbrowser_Taxonpage?taxid=482017
•
Barcode of Life (as Zatypota anomala) http://www.boldsystems.org/index.php/
Taxbrowser_Taxonpage?taxid=449222
•
Hymenoptera Online http://hol.osu.edu/index.html?id=50410
Nomenclature
This species was transfered from the genus Sinarachna to Zatypota by Zwakhals
(2006), but this act has not been consistently reflected by some online resources.
Hymenoptera Online lists this species under Sinarachna. BOLD has data under both
Sinarachna anomala and Zatypota anomala.
Material
a.
country: France; stateProvince: Bouches-du-Rhône; verbatimLocality: La Ciotat;
decimalLatitude: 43.198642; decimalLongitude: 5.631474;
coordinateUncertaintyInMeters: 30; samplingProtocol: in my semi-wild garden under a
lea
f of Pittosporum tobira, at about 1.3 m high, cocoon with wasp pupa and dead host
found 13 July 2012, cut leaf with cocoon kept outside, sheltered from sun and rain;
eventDate: 2012-07-20; individualCount: 1; sex: female; lifeStage: adult; catalogNumber:
RMNH.INS.593866; recordedBy: Hélène Dumas; associatedOccurrences:
RMNH.ARA.14254; institutionCode: RMNH; basisOfRecord: specimen
Dictynidae sp.
•
Barcode of Life http://www.boldsystems.org/index.php/TaxBrowser_TaxonPage?
subtaxa=hidden&taxid=1343
•
Araneae: Spiders of Europe http://www.araneae.unibe.ch/list/gen/taxId/17/Dictynidae
Material
a.
country: France; stateProvince: Bouches-du-Rhône; verbatimLocality: La Ciotat;
decimalLatitude: 43.198642; decimalLongitude: 5.631474;
coordinateUncertaintyInMeters: 30; samplingProtocol: in my semi-wild garden under a
lea
f of Pittosporum tobira, at about 1.3 m high, cocoon with wasp pupa and dead host
found 13 July 2012, cut leaf with cocoon kept outside, sheltered from sun and rain;
eventDate: 2012-07-13; individualCount: 1; sex: female; lifeStage: juvenile;
catalogNumber: RMNH.ARA.14254; recordedBy: Hélène Dumas;
associatedOccurrences: RMNH.INS.593866; associatedSequences: http://
www.boldsystems.org/index.php/MAS_DataRetrieval_OpenSequence?
selectedrecordid=3482013; institutionCode: RMNH; basisOfRecord: specimen
Spider hosts (Arachnida, Araneae) and wasp parasitoids (Insecta, Hymenoptera, ... 17

Notes
The host specimen could not be precisely identified using either morphology or
the Species Level Barcode Record search of the sequence library available on BOLD
at the time of this writing. A more general search of BOLD using the All Barcode
Records search option returned a closest match (95.26% similarity) with the dictynid
Nigma walckenaeri. A query of Genbank returned a closest match (88%) with Dictyna
latens. A calamistrum is visible on the fourth metatarsus of the presereved host
specimen (Fig. 17). The calamistrum is an organ involved in the spinning of cribellate
silk (Köhler and Vollrath 1995, Opell 1998). The presence of a cribellum alone
eliminates the vast majority of European spider taxa. In combination with the overall
size and shape, we conclude this host belongs to the spider family Dictynidae. The
World Ichneumonoidea database on Taxapad indicates that Zatypota anomala (Fig. 20)
has been associated with dictynid spider hosts (Yu et al. 2012). The incompletely
identified host DNA barcode sequence has been deposited in BOLD. As the library of
reference sequences grows, it may become possible to identify this host to species. An
attempt to barcode the parasitoid Zatypota anomala was not successful.
Discussion
DNA barcoding is best thought of as a supplement, not a replacement, for traditional
methods of taxonomic identification because both approaches have different, often
complementary strengths and limitations (Dayrat 2005, Will et al. 2005). There are now
many examples where the integration of DNA sequence data and morphological data have
a
dvanced knowledge in ways that would not have been possible without this synergy
(Riedel et al. 2013, Paquin and Hedin 2004). The study presented here is a case where
Figure 20.
Adult female Zatypota anomala (RMNH.INS.593866), specimen.
18 Miller J et al.

morphology alone is not adequate because of the physical condition and developmental
stage of the host at the time of death. Fortunately, we found that the condition of the host
specimens did not prevent the generation of DNA barcode sequence data.
Major advances in the study of host-parasitoid relationships require primary data from a
wide taxonomic and geographic range. Questions about host specificity and changes in
host-parasitoid relationships across large spatial scales are two important topics
hamstrung by the scarcity of primary data. But the slow pace of traditional approaches
means that few specialists can be dedicated to this area of study. These days, DNA
barcoding requires far less expertise, opening the door for more non-specialists to
contribute data in small quantities. Depositing these data online in community databases
like BOLD, and publishing results in an internet savvy journal dedicated to aggregating
datasets of all sizes, offers a strategy for advancing knowledge of host-parasitoid
relationships not available to previous generations of scientists.
Acknowledgements
Institutional funding and support provided by the Naturalis Biodiversity Center. Thanks to
Jan van Tol for administrative support, and to the Naturalis DNA Barcoding Facility for their
efficiency and professionalism. Special thanks to Hélène Dumas for contributing the
specimens from La Ciotat. Jose Fernandez-Triana (University of Guelph and Canadian
National Collection), Matjaz Kuntner (Slovenian Academy of Sciences and Arts), Pavel
Stoev (Bulgarian Academy of Sciences and Pensoft Publishers), and Lyubomir
Penev (Bulgarian Academy of Sciences and Pensoft Publishers) provided helpful
comments on a draft of the manuscript. Kees van Achterberg (Naturalis Researcher)
provided helpful guidance and advice.
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