DNA barcoding reveals diversity of Hymenoptera and the dominance of parasitoids in a sub-arctic environment.

0.95 MB·English

by  Stahlhut, Julie K

Stahlhutetal.BMCEcology2013,13:2
http://www.biomedcentral.com/1472-6785/13/2
RESEARCH ARTICLE Open Access
DNA barcoding reveals diversity of Hymenoptera
and the dominance of parasitoids in a sub-arctic
environment
Julie K Stahlhut1*, José Fernández-Triana1,4, Sarah J Adamowicz1, Matthias Buck5, Henri Goulet4, Paul DN Hebert1,
John T Huber3,4, Mark T Merilo1, Cory S Sheffield6, Thomas Woodcock2 and M Alex Smith1
Abstract
Background: Insect diversity typically declines withincreasing latitude, but previous studies have shown conflicting
latitude-richness gradients for some hymenopteran parasitoids. However,historical estimates ofinsect diversity and
species richness can be difficult to confirm or compare, because they maybe based upon dissimilar methods. As a
proxy for speciesidentification,weused DNA barcoding to identify molecular operationaltaxonomic units (MOTUs)
for 7870 Hymenoptera specimenscollectednear Churchill, Manitoba,from 2004 through 2010.
Results: We resolved 1630 MOTUs for this collection, of which 75%(1228) were ichneumonoids (Ichneumonidae +
Braconidae) and 91% (1484) were parasitoids. We estimate thetotal numberof Hymenoptera MOTUs inthis region
at2624-2840.
Conclusions: The diversity ofparasitoidsin this sub-Arctic environment implies a highdiversity ofpotentialhost
species throughout the same range. We discuss theseresults in thecontexts of resolving interspecificinteractions
that may include cryptic species,and developing reproducible methods to estimate and compare species richness
across sites and between surveys, especially when morphological specialists are not available to identify every
specimen.
Keywords: Barcoding biotas, Biodiversity, DNA barcoding, Hymenoptera, Sub-Arctic, Parasitoids,Canada
Background understanding of community biodiversity. Indeed, a
The region surrounding the town of Churchill, Manitoba, historic underestimation of biodiversity has undoubt-
lies at the intersection of the tundra and boreal forest edly hampered our understanding of Arctic food webs
biomes, making it an ideal site for investigating ecotone [6], while integrative approaches reveal previously
biodiversity. Previous studies of hymenopteran subtaxa unsuspected cryptic diversity within parasitoid-host
[1,2] and aquatic insects [3,4] have increased previous systems[7].
species richness estimates for this region by integrating Bymostmeasures,biodiversitygradientsgenerallyshow
morphological and molecular methods for species higher species richness at lower latitudes, but parasitoid
delimitation. Hymenoptera have been a commonly noted exception in
MembersoftheorderHymenopteraincludepollinators, some earlier literature, at least in comparisons of tropical
other herbivores, parasitoids, hyperparasitoids, predators, vs.temperateregions[8,9].Otherstudiessuggestthatthis
cleptoparasites, inquilines, and omnivores [5], so hymen- conclusion is incorrect, arguing that it is an artifact cre-
opteranspeciesplaymultipleandcomplexrolesinterres- atedbyerrorsofscale[10,11],overrepresentationofsome
trial food webs. However, accurate characterization studylocations[12],inconspicuousness or lowabundance
of trophic interactions is impossible without a better of some taxa [13], incomplete or inconsistent sampling
methods[12,14,15],andevenalatitudinalbiasinincorrect
taxonomy[16].
*Correspondence:[email protected]
1BiodiversityInstituteofOntario,UniversityofGuelph,Guelph,ON,Canada
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Stahlhutetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Stahlhutetal.BMCEcology2013,13:2 Page2of13
http://www.biomedcentral.com/1472-6785/13/2
The general pattern of reduced biodiversity at high Results
latitudes has been attributed to harsh climate [17,18], MOTUcounts
slower evolutionary rates due to long generation times For the overall Churchill collection (N = 7870), MOTU
[17], lossofpopulationsviadriftorinbreedingdepression counts calculated with jMOTU ranged from a high of
[17], and geographic/geological barriers to colonization 1898 (cutoff divergence = 1%) to a low of 1221 (cutoff
[19]. At the same time, other studies have found higher- divergence = 3%). For the Churchill 2010 subcollection,
than-expected diversity among Arctic and sub-Arctic MOTUcountsrangedfromahighof798(1%cutoff)to
Hymenoptera, both at our study site and in similar envi- a low of 660 (3% cutoff). The overall Churchill collec-
ronments. For example, a recent study of the boreal and tion and the 2010 subcollection are described in more
Arctic Microgastrinae (Braconidae) increased by 50% the detail in the Methods section. For brevity, we report
estimate of species richness for this subfamily in Canada remaining results for only the 2% cutoff unless other-
andAlaska[20]. wisespecified.
Inthecontextofsub-Arcticinsectecology,“expected” In the overall collection, we resolved 1630 MOTUs at
biodiversityisdifficulttodefine.Forexample,anthropo- the 2% cutoff level. The families represented by the
genic climate change may disproportionately affect most individuals – Ichneumonidae (n = 4797) and
high-latitude regions [2,21], making older diversity Braconidae(n=1367)–werealsothemostdiverse.We
estimates obsolete. Molecular methods for quantifying resolved 915 MOTUs for Ichneumonidae and 313
species richness typically exceed estimates made via MOTUs for Braconidae. The dominance of these two
morphology alone, because molecular methods both families was also evident in the 2010 subcollection, for
revealcrypticspeciesandpermitinclusionofspecimens whichweresolved722MOTUsatthe2%cutofflevel.This
that would otherwise remain unidentified [1,22]. subcollection included 1460 ichneumonids (461 MOTUs)
Comparisons among regions should be testable via and 288braconids (127 MOTUs). Ichneumonoids made
independent genetic and morphological evidence, but up 78% of the specimens in the full collection and 83%
the large scale of most ecological surveys make it of the 2010 subcollection. Ichneumonoid MOTUs also
impractical either to examine visible characteristics or made up 75% (= 228/1630) of overall MOTUs and 81%
tosequencemultiplegenesfromeveryspecimen[22]. (=588/722)ofthesubcollectionMOTUs.
We here report both the observed diversity and Along with the Ichneumonoidea, other parasitoids
the estimated richness for both parasitoid and non- dominated the collection. The superfamily Chalcidoidea
parasitoid Hymenoptera of the Churchill region, using accounted for 321 individuals (132 MOTUs) in the
abundance-based data from a comprehensive collection overall collection and 172 individuals (42 MOTUs) in
madebetween2004and2010.Wealsoassessedsimilar- the 2010 subcollection. The most diverse taxon outside
ity of assemblages among local collection sites, using the Ichneumonoidea and Chalcidoidea was another
incidence-based data from a subset of specimens parasitoid family – the family Diapriidae (Diaprioidea),
collected during the 2010 season. Our species counts with 163 individuals (83 MOTUs) in the overall collec-
and estimates are based on DNA barcodes [23], which tion and 80 individuals (45 MOTUs) in the 2010
we assigned to molecular operational taxonomic units subcollection.
(MOTUs)[24].Weusedthesedatatoconstructrarefac- Amongnon-parasitoids,themostnumerousanddiverse
tion curves, investigate diversity of subtaxa, and esti- family was the Tenthredinidae, with 479 individuals (72
mate species richness for Hymenoptera of the Churchill MOTUs) in the overall collection and 63 individuals (23
region. This approach has several advantages. First, it MOTUs)inthe2010subcollection.Thisfamilyaccounted
providesaproxyforspecies-leveldeterminations,which for all of the Symphyta in the Churchill collection, except
were not available for the entire collection (and rarely for seven individuals of a single species (Trichiosoma
are available for collections of this size). Second, it triangulum) of the family Cimbicidae (Tenthredinoidea),
reveals suspected cryptic species that can be further whichwerepresentintheoverallcollection.
studied and eventually described if their existence is Full specimen and MOTU counts for all represented
supported via integrative methods [25,26]. Third, this families and/or superfamilies of Hymenoptera are
approach can be standardized, which will make it easier summarizedinTable1.
for future researchers to compare results among similar
regionaldiversitystudies. Accumulation/rarefactioncurvesandrichnessestimates
We discuss our results in the context of overall Rarefaction curves for the complete Churchill collec-
hymenopteran diversity, the composition of different tion and the 2010 subcollection are shown in Figures 1
feeding guilds, and the potential of our approach to and 2. Neither set of curves approaches an asymptote
inform future studies of Arctic and sub-Arctic insect at the point where all individuals (Figure 1) or samples
communities. (Figure2)areincluded.
Stahlhutetal.BMCEcology2013,13:2 Page3of13
http://www.biomedcentral.com/1472-6785/13/2
Table1Specimencounts,feedingecology,andnumberofMOTU(2%divergence)foroverallChurchillcollectionand
2010subset
Superfamily Family Feedingecology N(all) MOTU(all) N(2010) MOTU(2010)
Apoidea Andrenidae Herbivore 5 3 0 0
Apidae Herbivore 82 8 0 0
Crabronidae Predator 7 6 0 0
Halictidae Herbivore 19 3 1 1
Megachilidae Herbivore 15 6 0 0
Ceraphronoidea Megaspilidae Parasitoid 2 1 1 1
Chalcidoidea – Parasitoid 321 132 172 42
Chrysidoidea Chrysididae Cleptoparasite 4 4 0 0
Dryinidae Parasitoid 18 3 9 2
Cynipoidea – Mixed/other 36 28 10 9
Diaprioidea Diapriidae Parasitoid 163 83 80 45
Ichneumonoidea Braconidae Parasitoid 1367 313 288 127
Ichneumonidae Parasitoid 4797 915 1460 461
Platygastroidea Platygastridae Parasitoid 40 29 5 3
Proctotrupoidea Proctotrupidae Parasitoid 23 8 19 6
Tenthredinoidea Cimbicidae Herbivore 7 1 0 0
Tenthredinidae Herbivore 479 72 63 23
Vespoideas.l. Formicidae Mixed/other 373 7 2 1
Pompilidae Predator 1 1 1 1
Vespidae Predator 111 7 0 0
TOTAL 7870 1630 2111 722
Fortheoverallcollection,theChao1richnessestimate Proportionalcontributionoftaxaandfeedingguilds
[27] was 2624, with a 95% CI range of 2446 through RelativecontributionstooverallMOTUdiversitywithin
2840. For the 2010 subcollection, the Chao 2 richness the overall Churchill collection are shown in Figure 4
estimate [28] was 1384, with a 95% CI range of 1233 (by taxon) and Figure 5 (by feeding guild). These
through 1578. Additional results for cutoff ranges from proportions remained similar throughout the 1-3%
1%through3%areshown inTable2. sequence divergence cutoff ranges for MOTU assign-
ments.ProportionsfortheChurchill 2010 subcollection
Speciessimilarityamongsites were similar to those for the overall collection (data not
Sobs [29,30] for each of the 12 sites in the 2010 sub- shown).
collection ranged from a low of 43 (site 8, 23 km SE) to
a high of 221 (site 2, 10 km SE). The largest estimated Discussion
shared species value was 413, between sites 5 and 12 Speciesidentificationanddelimitation
(12 km ESE and 9 km S), and the smallest was one, The 7870 specimens from the Churchill collection were
betweensites3and6(10kmSEand14kmE). assignedto1630MOTUsatthe2%cutoffrange.Onaver-
ThehighestvalueoftheChao-Sørensen-Estabundance- age, we identified a different species for every 4.8 indivi-
based similarity index [31] between any two sites was duals sampled (SD = 11.3). Most MOTU were sparsely
0.867, between sites 8 and 9 (23 km SE and 26 km SE, represented, with a mode of 1 (n = 734) and a median of
~ 5 km apart). The lowest similarity index was 0.020, 2.Only194MOTUsincluded10ormoreindividuals,and
betweensites6and 10(16km E and7 km ESE, ~ 9km only three of these included 100 or more (Figure 6). For
apart). There was no significant correlation between theChurchill2010subcollection,therewere722MOTUs,
similarity index and the distance between sites and the mean number of individuals per MOTU was 2.9
(p = 0.42, r = 0.10, df = 64; Figure 3). (SD = 4.5, mode = median = 1, maximum = 55, but
Full results (2% cutoff) for shared species and pre-sorting of this subcollection limited the number of
similarity are included in Additional file 1. Because replicates per MOTU). Singleton MOTUs made up a
Sobs for each site includes MOTUs shared with other similarproportionofthe2010subcollectionastheydidof
sites, the Sobs values for the 12 sites do not sum to the theparentcollection(362MOTUs=50%ofthe2010sub-
totalSobsforthe2010subcollection. collection;734MOTUs=45%oftheparentcollection).
Stahlhutetal.BMCEcology2013,13:2 Page4of13
http://www.biomedcentral.com/1472-6785/13/2
Table2EstimatedspeciesrichnessofChurchillHymenoptera,forMOTUcutoffvaluesof1%-through3%
Cutoff MOTU Chao1richness Chao195%CI Chao195%CI
lowerbound upperbound
1% 1898 3187 2977 3438
2% 1630 2624 2446 2840
3% 1221 1913 1770 2093
Cutoff MOTU Chao2richness Chao295%CI Chao295%CI
lowerbound upperbound
1% 798 1611 1436 1833
2% 722 1384 1233 1578
3% 660 1194 1067 1362
Uppertable:EntireChurchillHymenopteracollection(abundance-based,Chao1).Lowertable:Churchill2010subcollection(incidence-based,Chao2).
Based on number of MOTUs, the most diverse The second issue – unequal attention given to differ-
families in the overall Churchill collection were ent taxa – is not a trivial one, because full morpho-
the Ichneumonidae (915 MOTUs), Braconidae (313 logicalspeciesidentificationrequiresconsiderableeffort
MOTUs), Diapriidae (83 MOTUs) and Tenthredinidae and expertise, and identifying a diverse collection
(72 MOTUs). All of these families except the Diapriidae requires a diverse assemblage of specialists. The low
included some specimens that had been previously rate of morphological specimen identification says little
identified below the family level. We also resolved 132 about the ability of taxonomists to identify a specimen
MOTUsamongtheChalcidoidea,whichwerenotdeter- in hand, but much about the difficulty of placing every
mined to family. Each of the other taxonomic groups in specimen from a large survey into the hands of the ap-
this study, including the entire superfamily Cynipoidea, propriate specialist [23,34]. Specimens from this study
yieldedfewerthan30MOTUs. may receive more attention from specialists in the fu-
Asdetermined bytraditional morphologicaltaxonomy, ture, and the availability of DNA barcodes will make it
the 4787 specimens of Ichneumonidae in the overall col- easier for taxonomists to delimit and perhaps even de-
lection encompassed at least 13 subfamilies, 34 genera, scribe species in this collection via integrative methods
and 49 species. However, 1861 individuals (38.9%) car- [25,35].
ried no identification below the family level. At the 2%
cutoff level, 915 MOTUs were resolved for this family; if Rarefactionandrichnessestimates
this cutoff level provides a good approximation to inter- Rarefaction/accumulation curves show no clear ap-
specific divergence in Ichneumonidae, then morpho- proach to asymptotes for either the complete Churchill
logical examination previously resolved only 5.4% of the collection (abundance-based) or the 2010 subcollection
diversity present. Similarly, for 1367 specimensof Braco- (incidence-based over12sites) The Chao 1richnessesti-
nidae, 8.6% had no identification below the family level mate at 2% cutoff is 2624 MOTUs, with a 95% CI of
and the number of resolved species was only 40.9% of 2446 through 2840. By this estimate, the 1630 MOTUs
the MOTU total. For 479 Tenthredinidae specimens, defined from our collection cover approximately 62% ±
23.2% had no identification below the family level, and 5% of total richness. Since this estimate was based on
the resolved species count was 25.0% of the MOTU MOTUsrather than onmorphologicaldeterminations,it
total. isindependent oftheamountofattentioneachsubtaxon
These values should be considered approximations, receivedfrom amorphologicaltaxonomist.It shouldstill
for several reasons. First, no single MOTU cutoff be considered exploratory, because we did not sequence
level can definitively delimit all species; the 2% cutoff every one of the many thousands of individual speci-
level for MOTU definition is known to be insuffi- mens originally collected, and because the overall collec-
ciently stringent for some hymenopteran subtaxa and tion was not controlled for variation in sampling effort
too stringent for others [1,2,32,33]. Second, even the atdifferentlocations throughout the collectionrange.
better-identified families in this collection have not to Abundance-based measures may also be biased by
date received the same amount of scrutiny by mor- over- or under-representation of some species independ-
phological taxonomists, and the most completely ent of site selection or sampling effort. For example, our
determined taxa as of May 2012 (bees, vespid wasps, collection contained social insects (ants, bumble bees,
and microgastrine braconids) made up only a small and vespines) that can be oversampled when traps are
proportion of the collection. set near nests or other sites of high colony activity. As
Stahlhutetal.BMCEcology2013,13:2 Page5of13
http://www.biomedcentral.com/1472-6785/13/2
2000
1800
1600
1400
U
T 1200
O
M
of 1000
r
e
b
m
u 800
N
600
400
200
0
500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500
Subsample Size
1% 2% 3%
Figure1Abundance-basedrarefactioncurvesforentireChurchillHymenoptera2004-2010collection(N=7870),forMOTUcutoffvalues
of1%through3%.Dottedcurvesrepresent+95%CI(for1%cutoff)and-95%CI(for3%cutoff).
individual specimens, social Hymenoptera were among highly variable among different site pairs, ranging
the most abundant members of the collection, but also from 0.020 to 0.867. However, these values were not
among the least diverse, and this was verified by both significantly correlated with the linear distances be-
sequence data and morphological examination. Ants tween site pairs (Figure 2).
comprised 7 MOTUs, including Camponotus hercula- Almost all specimens in the Hymenoptera collection
neus (n = 27), two apparently distinct members of wereofwingedinsectsortheirlarvae,andweexpectthese
the Formica fusca complex (n = 45 and n = 282,) speciestodispersewidelyamongsites.Theonlyexceptions
1 2
one species of Myrmica (Myrmica alaskensis, n = 9,) wereworkerantsandfemaleDryinidae;however,sinceant
and up to three members of the genus Leptothorax reproductives and male dryinids are winged, these species
(n = 1, n = 4, n = 5). Similarly, the collection can still disperse via flight. The wide range of between-site
1 2 3
included seven identified species of Bombus whose similarities could be related to microhabitat differences,
sample sizes ranged from a minimum of two (B. fla- variationamongsamplingmethods,orsamplingatdifferent
vifrons) to a maximum of 44 (B. sylvicola), and times throughout the season. For example, even in the
which formed eight MOTUs (jMOTU split B. mixtus short sub-Arctic summer, theTrichoptera of the Churchill
and B. sylvicola each into two MOTUs, and pooled region show some seasonal variation in species presence
B. frigidus and B. jonellus into one MOTU). Three and abundance [4], and this may be true of Hymenoptera
species of Vespinae were also present, with sample as well. The Chao-Sørensen-Est similarity model should
sizes ranging from eight (Vespula intermedia) to 87 mitigatesomeoftheseeffects,sinceitincludesanestimate
(Dolichovespula albida). Vespine wasps were assigned of unseen species and also accounts for sample size differ-
to three MOTUs that were congruent with morpho- ence (the latter indirectly, via species/MOTU counts at
logical identifications. each pair of sites). Whether or not methodological or
microhabitat differences affected incidence or abundance
Similarityamongsites measuresofsomeMOTUs,the2010subcollectionshowed
For the 2010 subcollection, values of the Chao- noevidencefordistance-dependentsubstructureacrossthe
Sørensen-Est abundance-based similarity index were studyarea.
Stahlhutetal.BMCEcology2013,13:2 Page6of13
http://www.biomedcentral.com/1472-6785/13/2
900
800
700
600
U
T
O
M 500
of
r
be 400
m
u
N
300
200
100
0
0 2 4 6 8 10 12
Number of samples
1% 2% 3%
Figure2Incidence-basedrarefactioncurvesforChurchillHymenoptera2010subcollection(N=2111),forMOTUcutoffvaluesof1%
through3%.Dottedcurvesrepresent+95%CI(for1%cutoff)and-95%CI(for3%cutoff).
Feedingecologyandtheimportanceofparasitoids nymphal Hemimetabola, and arachnids [5,37,38]. At
The hymenopteran fauna of the Churchill region is least four ichneumonid subfamilies (Campopleginae,
dominated by parasitoids, and the parasitoid fauna of Ctenopelmatinae, Tersilochinae, and Tryphoninae)
Churchill is dominated by the Ichneumonoidea (Ichneu- target sawflies, and others (e.g. Mesochorinae) in-
monidae + Braconidae). We observed this pattern not clude hyperparasitoids. Non-ichneumonoid parasi-
only in the overall collection, but also in the 2010 sub- toids can also exploit a wide range of hosts,
collection, in which specimens were deliberately selected including Diptera (by Diapriidae), Hemiptera (by
for inclusion by a method designed to maximize taxo- Dryinidae), and the variety of hosts that are suscep-
nomic breadth. tible to attack by Chalcidoidea [5,37]. A more
Worldwide, the Ichneumonidae and Braconidae are detailed list of Churchill parasitoid subtaxa and their
thought to be the first and second most diverse families presumed hosts is included in Additional file 1.
of Hymenoptera [5,36], so this result is not unexpected Parallel studies of the Churchill fauna reveal no
in any regional survey of the order. Only about 12% of shortage of potential hosts. Our Hymenoptera collec-
identified parasitoid MOTUs were non-ichneumonoids, tion includes more than 70 presumed species of ten-
but theserepresentedatleast sixdifferenthymenopteran thredinoid sawflies. The comprehensive Churchill
superfamilies, including members of the families Diaprii- terrestrial arthropod collection includes approximately
dae (Diaprioidea), Dryinidae (Chrysidoidea), Megaspili- 1800 species of Diptera, 315 of Lepidoptera, 300 of
dae (Ceraphronoidea), Platygastridae (Platygastroidea), Coleoptera, 90 of Hemiptera, and 200 of Araneae [S.J.
Proctotrupidae (Proctotrupoidea), plus multiple families Adamowicz et al. pers. comm.]. These numbers are
ofChalcidoidea. probably conservative, since they are based on
Still, parasitoids cannot reproduce without hosts, observed MOTU or species richness rather than on
so the diversity of parasitoids in this sub-Arctic loca- extrapolated richness. If true species richness of these
tion implies a high level of host diversity. Many other orders is similar to that of Hymenoptera (e.g.
members of the superfamily Ichneumonoidea are approximately one-third of the true richness is not
parasitoids of immature Lepidoptera, but they also accounted for in the collection), then we would ex-
parasitize hosts from other holometabolous orders, pect that more than 4000 species from these groups
Stahlhutetal.BMCEcology2013,13:2 Page7of13
http://www.biomedcentral.com/1472-6785/13/2
1.000
0.900
0.800
0.700
x
e 0.600
d
n
y i 0.500
rit
a
mil 0.400
Si
0.300
0.200
0.100
0.000
0.00 5.00 10.00 15.00 20.00 25.00
Distance (km)
Figure3Chao-Sørensen-EstsimilarityindexforeachpairofsamplesintheChurchill2010subcollection.Thetrendlineisnotsignificant
(p=0.42,r=0.10,df=64).
are potential hosts for hymenopteran parasitoids in Geographyandclimate
the Churchill region. Details of host-parasitoid inter- Some previous surveys of regional ichneumonid fauna
actions are far beyond the scope of this survey, so we showed an unusual pattern – a higher diversity of
cannot tell whether some parasitoids attack multiple Ichneumonidae (but not necessarily other parasitoid
hosts, or whether some hosts are attacked by multiple taxa) in some temperate regions than in the tropics
parasitoids. However, DNA barcode data can provide [8,43]. However, this conclusion has been challenged
the earliest clues to the presence of cryptic host- [13,44], because in practice, it is difficult to design and
parasitoid interactions that can be targeted for future implement a sampling protocol free of methodological
taxonomic and ecological study [39,40]. bias. As a result, spurious patterns of diversity can be
Non-parasitoids in this collection were dominated traced to over- or under-sampling of some subtaxa or
by obligate herbivores, including bees (Andrenidae + habitats that share characteristics other than latitude
Apidae + Halictidae + Megachilidae, 20 MOTUs) and [12,13,45]. Some of these factors are additionally com-
sawflies (Tenthredinidae, 73 MOTUs). Predators were plicatedbyuncertaintaxonomy.Forexample,molecular
much less diverse (Crabronidae, six MOTUs, Pompili- evidence may reveal that some “generalist” parasitoid
dae, one MOTU, and Vespidae, seven MOTUs). species are actually made up of multiple cryptic species
Other aculeates included Chrysididae (four MOTUs) with more narrow host specializations than previously
and Formicidae (seven MOTUs). The chrysidids – all recognized[15,39,40].
members of the cleptoparasitic subfamily Chrysidinae – Theseissuesmakeitdifficulttodefine aprioriexpecta-
are likely to invade nests of host taxa that were tions of parasitoid diversity in a sub-Arctic environment,
represented in this collection [41]. One chrysidine, andtocompareitwithdiversityatotherlatitudes.Forex-
Omalus aeneus, is a known cleptoparasite in nests of ample, the earliest descriptions of anomalous latitudinal
Pemphredon spp. (Crabronidae), a genus represented gradientsinparasitoiddiversitypre-datetheroutineuse
by two individuals in the overall collection. The of both large-scale trapping [14,46] and molecular
other chrysidines were not identified to species, but methods [47,48] in biodiversity research. The MOTU
some Nearctic chrysidines are specialized cleptopara- approach is not always sufficient for species delimita-
sites of the cavity nests of eumenine vespids [41]. tion, and is not sufficient to describe new species, but it
The overall collection contained three determined has the advantage of being quantitative, reproducible,
species of cavity-nesting Eumeninae: Ancistrocerus and feasible even when morphological determinations
albophaleratus, A. waldenii, and Euodynerus leuco- are impractical [24]. As such, barcode-based diversity
melas [42]. studies can reveal large-scale geographic or ecological
Stahlhutetal.BMCEcology2013,13:2 Page8of13
http://www.biomedcentral.com/1472-6785/13/2
2000
1800
1600
1400
U
T 1200
O
M
of 1000
r
e
b
m
u 800
N
600
400
200
0
1 2 3
MOTU cutoff (% divergence)
Other Chalcidoidea
Apoidea Braconidae
Tenthredinidae Ichneumonidae
Diapriidae
Figure4RelativecontributionsbytaxontoMOTUdiversityinthecompleteChurchillHymenopteracollection.
patterns of cryptic diversity, and can also identify additional challenges of limited time and limited
specific taxa in need of further study by specialist taxo- resources. It is currently difficult to compare the diver-
nomistsorconservationbiologists. sityofthisregiontoeitherotherregionsoritsownpast,
becauseofthevariedmethodsusedinhistoricalstudies.
Conclusions DNA barcoding, combined with permanent curation of
The temptation to describe a regional fauna as un- the barcoded specimens, provides an avenue for rapid
expectedly diverse is often high, especially in surveys of estimation of species richness, as well as an easily stan-
extreme environments. The resolution of more than dardized method for designing and comparing future
1600 species of Hymenoptera in the Churchill region studiesandprioritizingcriticalresearch.
provides just such a case, because of both the harshness
of this environment and the dominance of parasitoids. Methods
The success of parasitoids is in turn a clue to the pre- Specimencollection,identification,andcuration
viously unexplored diversity of their hosts, especially Hymenoptera specimens were part of a larger arthropod
otherHolometabola. collection made in the Churchill area between July 2004
Fullspeciesdelimitationanddescriptionrequiremore and August 2010, inclusive. The majority of Hymenop-
detail than DNA barcoding alone can provide; these are tera specimens were collected via malaise trapping and
questions best answered by integrative methods that sweep netting. Latitude of collection sites ranged from
incorporate morphology, ecology, and genetics. How- 58.630° through 58.755°, and longitude from -93.819°
ever, in environments like that of sub-Arctic Canada – through-93.998°.
environments disproportionately threatened by climate We randomly sampled approximately 20% of the
change – taxonomists and ecologists have the twin entire Churchill Hymenoptera collection for barcoding
Stahlhutetal.BMCEcology2013,13:2 Page9of13
http://www.biomedcentral.com/1472-6785/13/2
2000
1800
1600
1400
U
OT1200
M
f
o1000
r
e
b
m
u 800
N
600
400
200
0
1 2 3
MOTU cutoff (% divergence)
Other/unknown Herbivores Parasitoids
Figure5RelativecontributionsbyfeedingguildtoMOTUdiversityinthecompleteChurchillHymenopteracollection.
(but see description below for the more detailed DNAextraction,amplificationandsequencing
selection protocol for specimens collected in 2010.) DNA was extracted from a single leg or abdomen of
Specimens were sorted to the order level at the Bio- each adult specimen, or a tissue sample from each im-
diversity Institute of Ontario (BIO, Guelph, ON, mature specimen, according to the methods of Ivanova
Canada) and at the Canadian National Collection of et al. [49]. Standardized primer sets [50,51] were used
Insects, Arachnids, and Nematodes (CNC, Ottawa, to amplify and sequence the 658-bp barcode region
ON, Canada). A total of 9562 specimens were sub- [23]. When amplification or sequencing failed, DNA
mitted to the Canadian Centre for DNA Barcoding was re-amplified and re-sequenced using primers that
(CCDB) laboratory at BIO for DNA isolation, amplifi- amplify smaller overlapping fragments and yield shorter
cation, and sequencing. Voucher specimens were sequences, but are still useful for specimen identifica-
retained at these locations after tissue was sampled tion and sequence analysis [52,53]. A list of the primers
for DNA analysis. Collection data, sequence data, used for barcoding of these specimens is provided in
and GenBank accession numbers for these specimens Additionalfile1.
are available through the Barcode of Life Data-
base (BOLD, http://www.boldsystems.org/), in the Datasetconstructionandverification
dataset DATASET-HYMCHUR1 (doi:10.5883/DATA- We constructed our primary dataset from the complete
SET-HYMCHUR1). More precise taxonomic determi- Churchill collection of Hymenoptera specimens that met
nations have been added for some specimens since the following criteria: (a) Collected between 2004 and
their initial identification, and further taxonomic 2010,inclusive;(b)Sequence≥200bp;(c)Sequencecheck
detail will be added to the BOLD dataset as work against BOLD ID engine consistent with existing ID or
progresses after publication. These updates will be image;(d)Nostopcodonsinsequence;(e)Fewerthan10
entered into BOLD and will become accessible ambiguous bases for sequences of length ≥ 350 bp, and
through this dataset. A summary of specimen records fewer than five ambiguities for shorter sequences; and (f)
and data, including GenBank accession numbers, is Sequence not previously published. Criteria (a) through
included in Additional file 1. (e) were used to exclude pseudogenes, contaminated
Stahlhutetal.BMCEcology2013,13:2 Page10of13
http://www.biomedcentral.com/1472-6785/13/2
800
700
600
U
T 500
O
M
of 400
r
e
b
m
u 300
N
200
100
0
1 2 3-5 6-10 11-20 21-50 51-100 > 100
Size of MOTU
Figure6DistributionofMOTUsizes(=individualsperMOTU)inthecompleteChurchill2004-2010collection.X-axisnottoscale,in
ordertoemphasizehighfrequenciesofsingletons,doubletons,andotherMOTUswithsmallnumbersofreplicates.
DNA samples, and low-quality sequences. Of the 9562 Of the 2111 specimens, 1257 were collected from mal-
specimens originally available, 7870 (82.3%) fit all criteria aise traps, 822 by sweep netting, 26 from pan traps, and
forinclusion.Atthetimeofdataanalysis,only1402ofthe sixbyothermethods.Specimensweresortedbycollection
7870 specimens (18%) had been identified to the level of eventandmorphospecies,withthegoalofcapturingmax-
species, provisional species, or species complex via imum diversity while minimizing redundancy. Beginning
morphology. So, for consistency, we subsequently refer to with the largest specimens from each event, we selected
specimens only by family (superfamily for the Cynipoidea 10 specimens per known parasitoid morphospecies and 5
andChalcidoidea).For7788specimens(99%),atleastone specimens per other morphospecies for molecular ana-
image is present in BOLD. There were two years of lysis. For some large and distinctive social insects (e.g.
particularly intensive study for Hymenoptera (2007 and Vespinae),fewerthan5individualswereincluded.
2010), while the full dataset included four specimens
collected in 2004, 88 in 2005, 887 in 2006, 3464 in 2007, Species/MOTUdelimitation
432in2008,457in2009,and2538in2010.Thecomplete Becausemanyindividualswerenotmorphologicallydeter-
2004-2010datasetwasusedtoestimatespeciescountand mined, we assigned each specimen to a MOTU using the
abundance-basedspeciesrichnessforHymenopteraofthe program jMOTU [54]. To reduce computational require-
Churchillregion. ments, we divided our overall data into subsets of < 1400
Wealsoassembledaseconddataset(asubsetofthefirst), sequences. Subsets were delimited by taxon, though not
designed to further investigate similarity of species assem- always at the same level (e.g. all chalcidoids were pooled
blages, and to estimate species richness via an incidence- inonesubset,whiletheIchneumonidaeweresplitintosix
based method. These sites were chosen because they subsets based on their cluster positions in a preliminary
yielded collections of comparable sizes, and all collections BOLD NJ tree). This method of partitioning the dataset
from these sites were sorted by the same researcher prevented accidental duplication of MOTU assignments
(MTM). The second dataset included 2111 specimens that across separate jMOTU runs. All jMOTU runs included
wereallcollectedbetween1Julyand15August2010,from MOTU cutoff definitions between 1 bp and 66 bp, cover-
12sitesthateachyielded50ormorespecimens(Figure7). ingarangeof0.15%to10%divergencewithrespecttothe
These sites span the range of habitats at Churchill, includ- full-length (658 bp) barcode. We used a low BLAST
ing fen, bog, boreal forest, tundra, and exposed Hudson identity filter of 95%, and a required sequence alignment
Bay shoreline (Additional file 1). Each one of these sites overlapof60%ofminimumsequencelength.
constituted one sample for the purpose of incidence-based For rarefaction/accumulation curves and estimates of
analyses. speciesrichness,weconstructedpreliminarydatafilesfrom