www.sciencemag.org/content/343/6176/page/suppl/DC1
Supplementary Materials for
Lethal Interactions Between Parasites and Prey Increase Niche
Diversity in a Tropical Community
Marty A. Condon,* Sonja J. Scheffer, Matthew L. Lewis, Robert Wharton,
Dean C. Adams, Andrew A. Forbes
*Corresponding author. E-mail: mcondon@cornellcollege.edu
Published 14 March 2014, Science 343, xxx (2014)
DOI: 10.1126/science.1245007
This PDF file includes
Materials and Methods
Figs. S1 to S9
Tables S1 to S10
References
1
MATERIALS AND METHODS
Organisms
Host-plants of Blepharoneura are functionally dioecious, highly sexually dimorphic vines
in the subtribe Guraniinae of the Cucurbitaceae (22). Male flowers are borne on a series
of inflorescences produced on actively climbing branches. Female flowers are borne at
the terminal nodes of pendulous branches (23). Sex ratios in populations of Gurania are
typically highly male-biased (15, 22). The male-biased sex ratio is a consequence of both
size-related sex expression and differences in flowering phenology of male-phase and
female-phase plants. Small vines are male and large vines are female (22); males usually
produce flowers continuously for several months, but female branches produce flowers
for ~7-10 days and then set fruit. Female branches can resume flowering anew after each
set of fruit mature. Male inflorescences bear multiple flowers; typically, just one flower
matures (opens) every other day. Flowers (both male and female) are open for just a few
hours on a single day, and then close. Closed male flowers of Gurania acuminata and G.
spinulosa (= G. lobata L.) abscise and fall to the ground the day after opening. Female
flowers generally remain attached to the plant.
Blepharoneura is a species-rich neotropical genus of tephritid fruit flies in the subfamily
Blepharoneurinae, which is the sister-group to the rest of the Tephritidae; all known hosts
of flies in the Blepharoneurinae are plants in the Cucurbitaceae (24). Most species of
Blepharoneura that have been analyzed genetically (15) are morphologically cryptic, but
have distinctive and highly complex courtship displays (25).
The Braconidae is one of the largest families in the Hymenoptera (26). Nearly all
braconid species are parasitoids (27). All braconid parasitoids of Blepharoneura are
members of the subfamily Opiinae, which includes 1981 named species (26). Bellopius is
a neotropical subgenus of Opius (28, 29), a large genus within the family Braconidae.
Prior to this work, Bellopius included 11 valid species. Hosts are known for only two of
these described species, and both attack flies in the family Tephritidae.
Sampling and Rearing
We collected flowers from inflorescences of two species of vines (Gurania acuminata, G.
spinulosa, Cucurbitaceae). Most of the readily accessible vines (N= 298) were growing
along the perimeter of a ~1km long airstrip at Los Amigos Biological Station (also
known as CICRA- Centro de Investigación y Capacitación Rio Los Amigos). The
airstrip’s location is defined by the following four corners: north-west 12°33'12.48"S 70°
6'30.42"W; north-east 12°33'12.21"S 70° 6'28.92"W; south-west 12°33'39.35"S 70°
6'17.38"W; south-east 12°33'38.46"S 70° 6'16.21"W. We also collected from three
female G. acuminata, five male G. acuminata, and four male G. spinulosa plants growing
within 1.5 km of the airstrip. We labeled and assigned a number to each branch bearing
inflorescences (Gurania acuminata: male branches N= 200, female branches N=3; G.
spinulosa: male branches N= 84, female branches N= 11), and recorded the precise
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location of each branch using GPS. From accessible plants around the airstrip, we picked
flowers daily from 4-16 October 2008. Fresh (pre-abscission) flowers that contain eggs or
larvae cannot be distinguished from un-infested flowers. To assess infestation rate, we
placed each flower in a separate, uniquely numbered clear plastic 1oz cup capped with a
tightly fitting lid, and recorded the species, sex, and maturation state of each flower. Cups
were checked daily for emergence of larvae and puparia. Puparia found in cups on 7-8
October, 11 October, 13 October, and 19-20 October, and a haphazardly chosen subset of
puparia found on 17 October, were placed directly in ethanol.
To rear remaining puparia to adulthood, we gently buried each puparium in moist media
in a separate individually labeled cup. Cups containing specimens (in all stages of
development) were transported in sealed cases carried with us in canoes on rivers in the
Amazon, and in baggage compartments of jets flying over the Andes and from Peru to
Iowa. Cups were held in facilities without climate control: the Los Amigos Biological
Station, hotel in Lima, and in USDA approved containment facilities at Cornell College,
Mount Vernon, Iowa. Cups were checked daily for emergence of adults. Most adults
emerged within 10-21 days. Adult braconid wasps, together with the puparia (“postemergence puparia”) from which they eclosed, were placed in labeled vials filled with
ethanol and frozen in a -80o freezer within a day of emergence. After one year in the
containment facility, puparia from which neither wasps nor flies emerged were
considered to be dead. We have no evidence suggesting that rearing conditions have
different effects on different species.
Molecular Methods
Extraction
We used the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA) to extract total
nucleic acids from all individuals in the study. For adult flies and wasps, genomic
extractions were performed using two legs from each specimen without maceration. Postemergence puparia were also extracted without maceration. This procedure allowed us to
preserve specimens for future morphological analyses. Pre-emergence puparia were
homogenized with disposable microfuge pestles and total nucleic acids purified using the
Qiagen Supplementary Protocol (“purification of total DNA from insects using the
DNeasy Blood & Tissue Kit”).
AFLP genotyping
We genotyped a panel of AFLP loci for 111 of the reared adult wasps. AFLP methods
could not be used for wasp DNA in pre-emergence puparia because Bellopius loci would
be intermixed with Blepharoneura loci. Genomic DNA from 111 adult Bellopius wasps
was digested with EcoRI and MseI (New England Biolabs, Ipswich, MD), and ligated to
cut-site specific adaptors (Applied Biosciences, Foster City, CA). Restriction-ligated
samples were then amplified using preselective primers Eco+C (5’GACTGCGTACCAATTCC-3’) and Mse+C (5’-GATGAGTCCTGAGTAAC-3’). Two
3
independent selective amplifications were performed on preselective amplicons with a
fluorescently labeled forward primer Eco+CAG (5’-GACTGCGTACCAATTCCAG-3’)
and each of two different reverse primers: Mse+CAA (5’GATGAGTCCTGAGTAACAA-3’) and Mse+CGA (5’GATGAGTCCTGAGTAACGA-3’). Selective amplification reactions were genotyped
on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). A panel of all
155 loci between 100 and 450 bp was assembled using GeneMarker v. 2.20 (Softgenetics,
State College, PA). Loci were first called automatically for all individuals, and then
checked visually. All samples were genotyped twice to ensure repeatability of AFLP
data. Loci that did not amplify in both genotyping runs for any single individual (<1% of
fragments) were discarded from the panel. Estimations of allele frequencies were
generated in the program AFLP-SURV (30) using a Bayesian method assuming a nonuniform prior distribution of allele frequencies (31). Estimated allele frequencies were
used to construct a 1-r distance matrix, which was then converted into a neighbor-joining
distance network using the programs NEIGHBOR and CONSENSE in PHYLIP 3.6 (32).
The network was bootstrapped using 10000 Nei’s genetic distance matrices (33) between
each cluster of individuals.
Sequencing
PCR amplifications were carried out with a Tetrad 2 thermocycler (Bio-Rad, Hercules,
CA, USA) with the following “touchdown” program: initial denaturation for 2 min at
92ºC, 12 touchdown cycles from 58ºC to 46ºC (10 s at 92ºC, 10 s at 58-46ºC, 1.5 min at
72ºC), 27 cycles at 10 s at 92ºC, 10 s at 45ºC, 1.5 min at 72ºC, and a final extension for 7
min at 72ºC. Primers for PCR and DNA sequencing are listed in table S1. PCR products
were cleaned for sequencing using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) or
gel purification using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA).
To identify selective priming sites that would amplify only fly or wasp mtCOI sequences,
we collected mtCOI sequences from reared (adult) opiinae parasitoids and compared
them to adult Blepharoneura sequences (15). Such comparisons allowed us to design
taxon-specific primers that exclusively amplify only Blepharoneura or opiine wasp
mtCOI even in the presence of the other. Opiine wasp-specific primers were designed
within the COI barcode region widely used for species identifications and in the Barcode
of Life Data System (34). Blepharoneura-specific primers were designed to amplify the
3’ end of mtCOI to allow comparison with previously identified Blepharoneura (15). The
same Blepharoneura-specific primers were used to PCR and sequence mtCOI for all flies
(adults, pre-emergence puparia, and post-emergence puparial exuviae of flies killed by
wasps) in this study.
We were careful to confirm amplification efficiency and specificity of primers by testing
with DNA from reared (adult) fly and wasp specimens. Wasp amplifications were also
confirmed by amplifying DNA from two sources for each adult specimen: DNA extracted
from adult wasps; and DNA extracted from the post-emergence puparial exuviae (“empty
puparia”) remaining after the adult wasp emerged. We could amplify both the fly and
4
wasp DNA from 100% of those empty puparia. Wasp sequences from empty puparia
matched sequences of DNA extracted from adult wasps that emerged from those puparia.
Sequencing reactions were carried out using Big Dye Terminator v3.1 Sequencing kits
(Applied Biosystems, Foster City, CA) and analyzed on an ABI 3730XL automated DNA
sequencer. Contigs were assembled for each gene region with the software package
Sequencher (Gene Codes Corp., Ann Arbor, MI). For all flies, we sequenced mtCOI
(504bp , N= 569 adults; 393 pre-emergence puparia; 163 post-emergence fly puparia that
yielded adult wasps). For all wasps (except figitids), we sequenced mtCOI (610 bp, N=
142 adults; N= 109 pre-emergence puparia). We also sequenced two nuclear genes from
adult Bellopius wasps: EF1-α copy F1 (excluding intron 1): 985bp (112 specimens); 28S
D2: 560bp (114 specimens). We used 28S D1-D3 region (1100bp) to identify adult
figitids. To identify figitids in pre-emergence puparia we developed a parasitoid specific
28S reverse primer to screen puparia (824 bp, D1-D2 region). All final contigs used in
the study have been deposited in GenBank (Accession nos. KF473465 - KF475237).
Wasp sequences will also be deposited in BOLD: Barcode of Life (34). Contigs were
assembled and aligned with Sequencher (Gene Codes Corp., Ann Arbor, MI). Alignment
of 28S was accomplished by eye. Alignments for mtCOI and EF1α were checked against
predicted amino acid sequences. Genetic diversity levels were determined by calculating
absolute and corrected P distances in PAUP* 4.0 (35).
Phylogenetic analysis of sequences: flies and wasps
Neighbor-joining (NJ) analyses and maximum parsimony (MP) analyses were conducted
in PAUP* 4.0b10 (35) using uncorrected “p” distances and treating gaps as missing data.
The percent bootstrap values were generated by analyzing 100 pseudoreplicated datasets
(with random input order of specimens for each replicate) using the neighbor-joining
method in PAUP*. A member of the Blepharoneura femoralis group (36) was used as an
outgroup for NJ analyses of the Blepharoneura mtCOI dataset. Bellopius bellus was used
as an outgroup for NJ and MP analyses of all braconid parasitoid datasets.
Species delimitation
Flies
To delimit Blepharoneura fly species, we used the same highly conservative criteria as
used previously with Blepharoneura: reciprocally monophyletic groups differing by at
least 4% mtCOI sequence divergence (15). To identify Blepharoneura species in our
sample, we used fly mtCOI sequences (N= 1107) from our sample of adult flies, preemergence puparia, and those post-emergence puparia from which wasps had emerged, to
construct a NJ tree (fig. S1) and to identify clusters of sequences >4% divergent from
other sequences. We compared our sample of Blepharoneura mtCOI sequences with
mtCOI sequences of flies representing 49 Blepharoneura species from other neotropical
sites (15).We discovered two previously undetected Blepharoneura species (nsp1 and
5
nsp2) and identified twelve Blepharoneura species previously found at other sites (fig.
S1, table S2).
Wasps
Our goal for this study was to discover patterns of parasitoids’ host-specificity. We did
not a priori assume that any of the parasitoids were specialists. Consequently, we used
several approaches that allowed us to identify distinct lineages (potential species)
independently of host-information. First, we used morphology to determine whether
wasps belonged to any recognized taxa. We then sorted female adult specimens into
“morphospecies”. Because many parasitoids are highly host-specific, best-practice for
parasitoid systematists is to use as much biological information as possible in the
diagnosis of species. To avoid such a priori bias toward recognition of specialists (albeit
often biologically justified), we carried out morphological analysis of adult wasps
without reference to host-information or genetic information. Because most surveys of
tropical parasitoid diversity are still largely based on morphological identifications (not
molecular evidence), our assessment of morphospecies diversity also allows comparison
with previous ecological surveys based solely on morphology, without molecular data (3,
37).
Molecular evidence often reveals morphologically cryptic species with distinct ecologies
(15, 19, 20). We used independent analysis of both morphological and molecular
evidence to determine whether any of the parasitoids attacking Blepharoneura are
members of cryptic species.
Morphology
Wharton identified adult wasps as members of two genera in the Braconidae (Opius and
Utetes) and one member of the Figitidae. Within Opius, two subgenera were recognized:
Thiemanastrepha and Bellopius. M. L. Buffington identified figitids and described them
as a new species based on molecular and morphological data (38).
To assess morphological diversity of Bellopius, Wharton first assessed morphological
variation of intact female specimens arranged in unit trays by specimen ID number,
without any reference to other collection data. Male specimens were excluded because
they usually lack morphological characters useful for separating species. Original
descriptions and keys to described species (39-41) provided an initial set of characters.
Most of those characters were invariant in the sample, leaving body color and ovipositor
length as the main criteria for sorting specimens into groups. Previous descriptions noted
major color differences among Bellopius species, but specimens from Los Amigos
included only a few specimens that differed in minor ways from the more uniformly
colored majority.
To verify ovipositor length differences, which were the most obvious morphological
differences among the Los Amigos specimens, Wharton and graduate student Lauren
Ward dissected ovipositors to measure the full length of the fused dorsal valves, the base
6
of which is nearly always concealed on intact specimens. Wharton also measured wings,
hind legs, and mesosomal length as a proxy for body size. Using ovipositor length and
mesosoma length, both as absolute values and as ratios of ovipositor to mesosoma length,
he then looked for gaps in distributions. Using those characters, he separated nine
morphospecies (table S3), two of which were supported primarily by minor color
differences. Morph 9 was further divided into three groups.
Molecular evidence from Los Amigos 2008 specimens
To rigorously evaluate species limits within our sample of Bellopius, we assessed
corroboration among analyses of four independent molecular datasets: AFLPs and DNA
sequence data from one mitochondrial and two nuclear genes. First, we used AFLPs to
analyze 155 independent loci from 111 adult wasps (fig. S2) to categorize Bellopius
groups into Molecular Operational Taxonomic Units (MOTUs) defined as reciprocally
monophyletic groups with > 50% bootstrap support in a neighbor joining network. We
identified 12 MOTUs representing provisional Bellopius species (spA, spB, spC, spD,
spE1, spE2, spF, spG, spI [a singleton], spK, spL, & spM; fig. S2). AFLP-defined MOTU
groups are especially important because they reflect relationships revealed by many loci
within the nuclear genome.
We then tested AFLP-based MOTUs for congruence with mtCOI sequences. We
considered mtCOI divergence of 1% or greater between AFLP-defined MOTUs to be
strong independent corroborating evidence for provisional species (table S4). We chose
the 1% criterion as an arbitrary, but comparatively conservative criterion. Previous work
using total evidence (including patterns of host-specificity, which we explicitly ignore)
recognized cryptic species of skipper butterflies differing by as little as 0.32% mtCOI
divergence (42), and recognized cryptic species within a group of braconid parasitoids
that showed an average of only 0.15% sequence divergence (19). Our 1% threshold was
met by seven Bellopius MOTUs (spA, spB, spC, spD, spF, spG, spI) and identified two
additional singletons (spH and spJ) not present in the AFLP dataset because they were
represented only by pre-emergence puparia (fig. S3). The following sets of species shared
no mtCOI haplotypes, but included some mtCOI sequence divergences below our 1%
threshold: Bellopius spp E1 and E2 (0.8-1.3%); spM, spK, and L (0.6-1.3%).
To find out if nuclear sequences corroborated patterns revealed by AFLP loci and mtCOI,
we constructed both neighbor-joining (NJ) trees and maximum parsimony (MP) trees for
the nuclear genes ef1-α and 28S. Because AFLP and nuclear data are available only from
adult specimens, singleton Bellopius species H and J (represented only in pre-emergence
puparia) are omitted from these analyses.
Trees generated from both nuclear genes have very short branch lengths, suggesting that
nuclear genes ef1-α and 28S D evolve more slowly than mtCOI; such differences in rates
of evolution of mtCOI and nuclear genes are not uncommon (15, 43-45). Nevertheless,
trees supported 7 MOTUs: spA, spB, spC, spD, spE2, spI [singleton], and spM. Bellopius
spF was supported by the 28S tree but no ef1α sequence data were available for spF.
Because branch lengths in NJ trees were short, we generated MP trees with numbers of
7
base-pair differences plotted on branches because MP trees more clearly show
relationships between individual DNA sequences found in each MOTU (figs. S4, S5).
Relationships between MOTUs in NJ and MP trees were identical in topology. In the ef1α tree (fig. S4), MOTUs spC and spD were not reciprocally monophyletic, in contrast to
the AFLP and mitochondrial results; however, in the 28S tree (fig. S5) spC did form a
clade independent of all spD individuals. Two pairs of provisional species, spE1/spG and
spL/spK shared identical 28S and ef1α sequences. Placement of spE1 in a separate clade
from spE2 supports our decision to recognize those lineages as distinct provisional
species. Although slowly evolving nuclear genes failed to separate spE1/spG and
spL/spK, AFLP data representing 155 independent nuclear loci support monophyly of
each of those four provisional species, and we found no shared mtCOI haplotypes among
those four groups.
We used mtCOI sequences to generate NJ trees for Thiemanastrepha wasps (fig. S6).
Thiemanastrepha mtCOI grouped into two discrete clusters differing by 4.4% sequence
divergence. For the purposes of this paper, we call these provisional species T. spA and
T. spB. We found only one Utetes species; the two specimens we collected of this genus
were genetically identical at mtCOI. All figitid parasitoids had identical 28S D1-D3
sequences and have been described as a single species (Tropideucoila blepharoneurae).
Results of molecular analysis of figitids are published elsewhere (38).
Molecular evidence (mtCOI) from broad geographic sample of wasps
Because members of two pairs of lineages (L/K and E1/G) shared identical nuclear
sequences, we sought additional evidence to increase our confidence that the Bellopius
parasitoids from Los Amigos (identified by AFLP, mtCOI, and two nuclear genes)
represent distinct lineages worthy of provisional species status. We compared mtCOI
sequences with a sample of wasps reared to adulthood from flies collected at diverse
geographic sites (table S2) or during a different year (2007) at Los Amigos (fig. S7).
Collections from geographically distant sites (100- 3500 km) included specimens of
Bellopius with mtCOI haplotypes identical or nearly identical to Bellopius spp A, B, C, F,
H, I, L and M (fig. S7). Bellopius spp H, I, and J were represented by single specimens
(singletons) in our 2008 Los Amigos sample, but clearly group with specimens either
found elsewhere (spH, spI) or at Los Amigos in 2007 (spJ). Five species were not found
at other sites: Bellopius spp D, E1 (narrowly defined as E1b, see fig. S7), E2, G, and K.
All provisional species (with the exception of the K/L/M group) were supported by
bootstrap values > 98%. The K/L/M group (77% bootstrap support) includes two lineages
with 100% bootstrap support: spM and lineage K/L. The K/L lineage includes
provisional spK (81% bootstrap support) and spL (55% bootstrap support).
Bellopius “spE1” (n=5) was the only species of Bellopius at Los Amigos for which adult
wasp and puparial samples revealed non-overlapping patterns of parasitoid- fly
associations (Fig. 3). Comparison with adults from diverse localities (fig. S7) suggests
that Bellopius “spE1” is actually a complex of recently diverged host-specific lineages:
three monophyletic groups- species E1a, E1b, E1d) and one “group” (labelled E1c) with
no bootstrap support. Bellopius spE1b includes three specimens, all from Los Amigos,
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all reared from flies found in male flowers of Gurania acuminata (two adults pBpup537,
532 reared from fly sp28; one- pBpup416- from pre-emergence puparium of fly sp3).
Bellopius spE1a is represented by two adult specimens, both reared from female flowers
of G. spinulosa, one from Los Amigos in 2007 (Bwsp778 ex. fly sp30) and one from the
Pto. Maldonado-Infierno transect in Peru (Bwsp753 Pe05). Bellopius spE1d is
represented by two adult specimens reared from male flowers of G. spinulosa, one from
Los Amigos in 2007 (Bwsp774 ex. fly sp12) and one from French Guiana (Bwsp1166).
We suspect that specimens labeled as “E1c” do not represent a single species, but we are
unable to place them with confidence given our currently limited geographic sample. All
of the E1c specimens emerged from flies in male flowers of G. spinulosa: two are adults
from the Guianan Shield (Bwsp77-Suriname, Bwsp1118-French Guiana) and two
specimens are from ethanol-preserved pre-emergence puparia from our Los Amigos 2008
collection (pBup68 in puparium of fly sp30 and pBpup276 in puparium of fly sp8).
Comparison of morphological and molecular evidence from wasps
We compared Bellopius morpho-species groups with MOTU provisional species (table
S5). The three Bellopius singleton MOTUs (H, I, J) were not represented in the
morphological sample: spH and spJ were only found in pre-emergence puparia, and spI
was represented by a male specimen. Three MOTU species (Bellopius spp E1, E2, and F)
formed clearly morphologically distinct groups, each with increasingly long ovipositors.
Thus, morphological evidence (though based on a single female specimen) provides
further support for Bellopius E1 as a provisional species distinct from spG.
Morphological evidence also sheds some light on the Bellopius spp K, L, and M groups.
All spK analyzed morphologically (N=4) fell into Morph 9c, together with 13 of 17
specimens of spM, and two of five specimens of spL (tables S4, S5), providing additional
evidence that the three lineages are closely related, but show different patterns of
morphological variation.
Most MOTU species fell into morphological groups that included members of other
MOTU (molecular) lineages. All Bellopius MOTU sp D clustered together in Morph8;
however, Morph8 also includes an individual of MOTU spG. MOTU spC (N=1) was
placed in Morph5, but Morph5 also includes multiple MOTU spG individuals. All
Bellopius MOTU spK clustered in Morph9c along with individuals of MOTU spA, B, L,
and M. Bellopius MOTU species A sorted into two Morph9 groups (9b, 9c) and spB fell
into three separate morphological groups (Morph4, 6, and 9c) based on color and
ovipositor length. Individuals of MOTU spM were sorted into four groups (Morph4, 7,
9a, 9c), and spL was placed into two groups (Morph4, 9c, which both included spM).
This preliminary morphological assessment suggests that all but three of the eleven
species of Bellopius included in morphological analyses are cryptic species. Based on our
experience with morphologically cryptic species of flies (25) we expect that future
morphological analysis of new character-sets (using morphometrics and other methods)
will reveal morphological differences among these Bellopius species.
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Summary of wasp species delimitation
We find compelling support for 14 provisional species of Bellopius at Los Amigos. Table
S6 summarizes the different independent types of evidence used to define provisional
species representing distinct genetic lineages that maintain their distinction in sympatry.
Our provisional species assignments are consistent with the “unified species concept”
(46). AFLPs represent a coarse but informative snapshot of genome-wide divergence
(155 independent loci) and reveal clearly differentiated sympatric clusters. Importantly,
AFLP-based MOTUs were checked with information from single gene sequences (COI,
28S, and ef1α). Evidence from mtDNA provides strong support for our hypothesis of 14
provisional Bellopius species. Nuclear data (with the exception of the E1/G group) are
also consistent with AFLP/mtCOI data: the more slowly evolving nuclear genes do not
distinguish more recently diverged lineages (e.g., K/L/M). Only the nuclear genes failed
to distinguish Bellopius spE1 and G, but all other molecular data (figs. S2, S3, S7) and
morphological data (tables S4, S5) strongly support E1 and G as distinct groups.
Had we included ecological data to identify provisional parasitoid species, as is not only
common but also “best practice” among parasitoid systematists (19, 29, 47), our
ecological data would further support these fourteen provisional species. All Bellopius
species (except K/L) are lethal to just one species of Blepharoneura. Furthermore,
Bellopius spK and L, which appear to be quite closely related and overlap in their
patterns of host-use (Fig. 3), nonetheless differ significantly in the proportions of each
host-fly attacked (Fisher’s exact test: successful adult wasps p= 0.0005; adults and preemergence puparia: p< 0.0001).
Ecological patterns
We evaluated patterns of host-plant-part infestation by counting the number of flowers in
which we found at least one puparium (Fig. 1). Most flowers were not infested. Of the
flowers that were infested, most yielded a single puparium, but a few yielded as many as
four or five (table S7). These patterns of infestation are typical in this system, even for
very rare host-plants (15, 25).
Using species identified by molecular methods (see Species Delimitation), we evaluated
patterns of species abundance and rates of parasitism (fig. S8) and host-specificity (Figs.
2-4; figs. S2-S6, S9). We tested the null hypothesis that patterns of associations (Fig. 3)
between wasps and fly-hosts revealed by adult versus pre-emergence puparia are the
same. This hypothesis was evaluated both by a Fisher’s Exact Test (table S8), as well as a
resampling procedure (fig. S10). For the latter, we carried out a permutation test. We
simulated adult distributions from those observed in pre-emergence puparia. We asked:
do wasp-fly associations differ between adult wasps and pre-emergence puparia? To
answer that question, we simulated patterns observed in adults, using the observed
frequencies of associations between wasps and flies in pre-emergence puparia. This
method tests the null hypothesis that the wasp-fly associations do not differ between adult
wasps and pre-emergence puparia, while taking the observed frequencies of wasps and
10
sample sizes into account. The observed odds-ratio of adult wasps and pre-emergence
puparia was obtained from the observed data. Next, the adult wasp data were resampled
(with replacement) in proportion to the observed frequencies of wasp-fly associations in
our pre-emergence sample. We obtained an odds-ratio for these data. This process was
repeated 9,999 times to generate a distribution of expected patterns relative to the
frequency and sample sizes of pupae, from which the observed pattern of adult wasps
was compared.
Based on both statistical procedures we reject the hypothesis. Specifically, Fisher’s exact
test revealed a significant difference in the proportion of each host-fly attacked (P =
0.0075), which was confirmed by the resampling distribution (Odds = 0.0458; Prand =
0.0001: fig S10). Thus, we see a distinct pattern: all but one species of wasp (10/11,
excluding singletons) emerges from just one species of fly, but puparia reveal that the
majority of wasp species actually attack >1 species of fly.
We also evaluated patterns of “mistakes” made by Bellopius parasitoids. We consider
“mistakes” to be vulnerable Bellopius (i.e., those Bellopius that lay eggs in a species of
Blepharoneura but never emerge as adults). To assess patterns of mistakes, we first
evaluated the number of flower-bearing branches that bore flowers infested with
Blepharoneura (table S9). Two Blepharoneura species was the average number of fly
species associated with branches bearing female flowers of both species, and male
flowers of G. acuminata. Three Blepharoneura species was the average number of fly
species found on branches of G. spinulosa bearing male flowers (table S9). For each
Bellopius parasitoid that made a mistake, we determined whether the mistake was made
on branches with flowers infested by the “correct” species of Blepharoneura (table S10).
Patterns are striking: all but one wasp (a member of the problematic “E1c” group; fig. S7)
made a mistake only when the “correct” species of Blepharoneura was present (“correct
species” = a species of fly from which an adult wasp can emerge). That one wasp
(pBpup276), which we scored as the single “exception to the rule” (i.e., the “rule” that
mistakes are made only when the “correct” host is present; table S10), actually may not
be an exception. The wasp (pBpup276) recovered in a fly sp8 puparium belongs to the
problematic lineage Bellopius spE1c, not to spE1b (fig. S7). The “correct” host of spE1b
is Blepharoneura sp28, which lays its eggs exclusively in male flowers of G. acuminata,
but pBpup276 was reared from a male flower of G. spinulosa (table S10). Although we
do not yet have data on susceptible Blepharoneura hosts for “E1c”, we identified
Bellopius spE1c in a pre-emergence puparium of Blepharoneura sp30, a widespread
species that mainly attacks male flowers of G. spinulosa (15). If fly sp30 is the “correct”
host of Bellopius spE1c, then its “mistake” (like the mistakes made by all other wasps)
did occur on a branch with its “correct” host. Five specimens of sp30 were reared from
male flowers on a single branch (branch 29) of G. spinulosa (tables S9, S10).
11
fig. S1. Left: Neighbor-joining (NJ) tree of Blepharoneura fly species identified by
mitochondrial cytochrome oxidase (mtCOI) gene (504 bp) sequences matching
previously identified species (15) –or appearing as species apparently endemic to the Los
Amigos region (nsp1, nsp2). Colors associated with Blepharoneura species labels
correspond to colors identifying fly-hosts of wasps in Figs. 2-4. Branch tips bear labels of
“placer specimens”: blue m= male flower, red f= female flower, gold GA= Gurania
acuminata, purple GS= G. spinulosa; Pe08 = representative specimen from our Los
Amigos collection. Other sites (table S2): Bo= Bolivia; EcB= Ecuador- Bilsa Biological
Station (western Ecuador); EcJS= Ecuador, Jatun Sacha Biological Station (eastern
Ecuador); FG= French Guiana; Pe87= Explorer’s Inn, Tambopata National Reserve,
Peru. Right: Pie charts show patterns of host-plant use by Blepharoneura at Los Amigos.
Sample size (our Los Amigos collection) is indicated to the right of each pie chart.
Blepharoneura species’ geographic distributions are indicated by letters associated with
previously sampled sites (15). Note that “P” represents a roadside transect between Pto.
Maldonado and Infierno in Madre Dios, Peru (table S2) not the Los Amigos site.
Bootstrap values are indicated above branches.
12
fig. S2. AFLP network based on analysis of 155 independent loci for 111 adult Bellopius
wasps reared from Blepharoneura flies. Colors denote host fly species (as in Figs. 2-4
and fig. S1). Numbers at nodes represent bootstrap support of >50%.
13
fig. S3: Neighbor joining (NJ) tree for Bellopius wasps based on mtCOI sequences (610
bp). Bellopius species identified using AFLP loci (fig. S2) are indicated by vertical bars
and boldface letters. Colors in specimen labels correspond to host-plant sex (blue m=
male flower, red f= female flower), host-plant species (gold= Gurania acuminata; purple
= G. spinulosa), and host-fly species (colors as in Figs. 2-4 and fig. S1.). Samples with a
picture of a puparium represent DNA extracted from ethanol-preserved pre-emergence
puparia. Samples with a picture of a wasp represent DNA extracted from adult wasps that
emerged from Blepharoneura puparia. Bootstrap values are indicated above branches.
14
15
fig. S4. Maximum parsimony (MP) tree for adult Bellopius wasps based on nuclear gene
ef1-alpha copy F1 sequences (1006 bp). Color coding and notation of specimens as in fig.
S3; adult/puparium icons are absent (only adult specimens are included). Numbers above
branches represent number of bp distinguishing groups.
16
17
fig. S5. MP tree for Bellopius wasps based on nuclear gene 28S D2 sequences (560bp).
Color coding and notation as in figs. S3-S4. Numbers above branches represent number
of bp distinguishing groups.
18
fig. S6. Neighbor joining (NJ) tree for Thiemanestrepha wasps based on mtCOI
sequences (610 bp). Color coding and notation as in figs. S3-S5. Bootstrap values are
indicated above branches.
19
fig. S7. NJ tree based on mtCOI sequences (610 bp) showing relationships of Bellopius
from Los Amigos (star) to Bellopius adults reared from fly pupae from Gurania flowers
at other localities (fig. S1, table S2) and wasps collected at Los Amigos in 2007 (Pe07).
Triangle = Eastern Ecuador (Napo), circle = Suriname; square= French Guiana; line =
Bolivia (Sta. Cruz to Villa Tunari); diamond = Peru (Pto. Maldonado to Infierno). Los
Amigos “singletons”, two represented only by puparia (spH, J), all group with wasps
collected on different dates or at different sites. Shaded inset: Geographic patterns also
reveal patterns within “E1” suggesting that the lineage (an apparent ecological anomaly
in our Peru08 collection) may actually include at least four species. Five of the 14
Bellopius species have only been collected at Los Amigos: D, G, K, E2, and E1b.
Bootstrap values are indicated above branches. Color coding and notation as in figs. S1
(localities), S3-S6 (wasp specimens and hosts).
20
fig. S8a,b. Fly abundance and patterns of parasitism by Bellopius: A) abundance of
Bellopius parasitoids is positively correlated with host-fly abundance (Pearson’s r =0.58).
B) Percent of flies attacked is not related to host-fly abundance (Pearson’s r = -0.05).
Colors correspond to host-flies (Figs. 2-4; figs. S2-S7).
21
fig. S9. Most species of Blepharoneura are killed by a single species of Bellopius. Pie
charts (N= flies killed by Bellopius; i.e. the number of adult wasps reared from each
Blepharoneura species) represent the proportion of each Blepharoneura killed by
particular Bellopius species. Species of lethal Bellopius are indicated by letters either
inside or at the periphery of the pie chart. Letters correspond to provisional Bellopius
species (Figs. 3,4; figs. S2, S3).
22
fig. S10. Histogram of odd-ratios for adult wasps and puparial (pre-adult emergence)
wasp data obtained from resampling procedure. The observed value (0.0458) is denoted
by the asterisk, and is highly significant (Prand = 0.0001).
*
Randomly generated odds-ratios
23
table S1: Primers used for PCR amplification (*) and DNA sequencing. Most primers
were designed for this study (**).
Gene
Primer
Primer sequence 5’-3’
Source
fly-COI
BpupCOIF*
BpupCOIR*
BwspCOIF*
BwspCOIR*
rc28A*
HYM28SD2F
HYM28SD3R
28C*
Bwsp28SD2selR2
BelEF40F*
BelEF46F
BelEF61AR
BelEF53R*
TAGGAATAATYTAYGCAATAATRGCAATTG
GAAGANCCAATWGTTGARATWACRTTTCAAG
GTTTATCWATAAGAWTAATTATTCG
CTTTCATTAWAAATAATATGAGA
AGCGGAGGAAAAGAAAC
CGTGTTGCTTGATAGTGCAGC
TCGGAAGGAACCAGCTACTA
GCTATCCTGAGGGAAACTTCGG
GGTCCTGAAAGTACCCAAAGC
GAGAAGGAGGCGCAGGAGAATG
CGAAGAAATCAAGAAGGAAG
GAYGCTGGGCTGTAGCCRATCTTC
GTGAGCAGTATGACAATCCAAAACAG
**
**
**
**
(48)
(49)
(49)
(48)
**
**
**
**
**
wasp-COI
28S Dloop
Ef1a
24
table S2: Los Amigos Blepharoneura species also identified at other geographic localities.
Blepharoneura
species
Sp1
Sp2
Sp3
Sp4
Sp8
Sp10
Sp11
Sp12
Sp21
Sp28
Sp30
Sp44
Localities where species were previously identified (15)
Bolivia (Sta. Cruz- Villa Tunari); French Guiana (Cayenne to St. Georges)
Bolivia (Sta. Cruz- Villa Tunari); Peru (Pto. Maldonado to Infierno);
Peru (Pto. Maldonado to Infierno)
Bolivia (Sta. Cruz- Villa Tunari); Ecuador- Jatun Sacha (Napo); French Guiana (Cayenne to St. Georges); Peru (Pto.
Maldonado to Infierno); Venezuela (Guatopo National Park)
Bolivia (Sta. Cruz- Villa Tunari); Ecuador- Jatun Sacha (Napo); French Guiana (Cayenne to St. Georges)
Bolivia (Sta. Cruz- Villa Tunari); Ecuador- Jatun Sacha (Napo); Ecuador- Bilsa (Esmeraldas); French Guiana (Cayenne
to St. Georges); Peru (Pto. Maldonado to Infierno); Venezuela (Guatopo National Park)
Ecuador- Jatun Sacha (Napo); French Guiana (Cayenne to St. Georges); Peru (Pto. Maldonado to Infierno)
Ecuador- Jatun Sacha (Napo)
Bolivia (Sta. Cruz- Villa Tunari); Ecuador- Bilsa (Esmeraldas); French Guiana (Cayenne to St. Georges); Peru (Pto.
Maldonado to Infierno); Venezuela (Guatopo National Park)
Bolivia (Sta. Cruz- Villa Tunari); Peru (Pto. Maldonado to Infierno)
Bolivia (Sta. Cruz- Villa Tunari); Ecuador- Bilsa (Esmeraldas); French Guiana (Cayenne to St. Georges); Peru (Pto.
Maldonado to Infierno); Venezuela (Guatopo National Park)
Peru (Explorer’s Inn, Tambopata National Reserve)
25
table S3: Morphological analysis of Bellopius identified nine provisional species groups
(“morphs”).
Morph
group
Morph1
Morph2
Morph3
Morph6
Size ratios
ovip/mesosoma
very long
> 2.3
long
> 2.3
long, slightly longer 1.85-1.95
than Morph2
short
1.21-1.32
long (= Morph3,
1.5-1.6 (except
except bwsp533 a
1.41 for
bit shorter)
bwsp533)
medium
1.5
Morph7
medium
Morph4
Morph5
Ovipositor traits
Morph8
long (= Morph2)
Morph9 (three subgroups)
Morph9a low end of medium
range
Morph9b low end of medium
range
Morph9c medium
Color
body size
pale
pale
pale
large
very, very small
moderate
pale
pale
moderate
large
smaller than
Morph3 & 4
Very small
1.5-1.6
somewhat darker
on abdomen
somewhat darker
on abdomen
pale
pale
pale
1.4-1.45
pale
1.35-1.45
pale
Low end of
medium range
moderate
1.65
1.65-1.75
26
moderate
small (= Morph6)
table S4: Pairwise sequence divergences between mtCOI sequences from adult specimens of the provisional species identified using
AFLPs. The same 111 adult wasps are used in this comparison as were used in AFLP work.
A (n=3)
B (n=4)
C (n=4)
D (n=8)
E1 (n=2)
E2 (n=8)
F (n=2)
G
(n=19)
I (n=1)
K
(n=11)
L (n=12)
M
(n=37)
A
0
1.92.1
2.93.0
3
5.35.5
5.15.5
5.3
4.54.8
4.54.6
5.15.5
5.5
4.95.3
B
C
D
E1
E2
F
G
I
K
L
M
0.0-0.2
3.8-4.3
0
3.8-4.0 1.4-1.5
0
6.0-6.4 6.4-6.6 5.8-6.0
0.0-0.2
5.9-6.6 6.1-6.6 5.7-6.0
0.8-1.3
0.0-0.9
6.4-6.6
5.6-6.0
6.4
6.2
5.8
5.6-5.8
2.8-3.2
4.5-5.0
2.7-3.2 0.0-0.2
4.4-4.8 3.6-3.8 0.0-0.2
5.6-5.8
5.6
4.8-4.9
4.1-4.3
3.9-4.3
5.8-6.2 5.5-5.8 5.2-5.7
4.1-4.4
4.0-4.7 5.1-5.3 5.5-5.8 2.8-3.1 0.0-0.8
6.2-6.4 5.3-5.6 5.0-5.3
5.3-5.8 5.8-6.2 5.6-6.2
4.1-4.3
4.9-5.3
4.2-4.7
5.1
5.6-6.0
3
0.6-0.8
0
3.9-4.7 4.9-5.3 5.1-5.6 2.8-3.2 0.6-1.3 0.9-1.3 0.0-0.8
4.3
27
4.1
0
table S5: Comparison of morphologically (Morph group, table S3) and molecularly
defined (MOTU) species groups (figs. S2, S3). Specimen numbers listed on the same line
are members of the same MOTU group. Colors, boldface, and underlining provided to
show match between specimen numbers and MOTU groups
MOTU
Morph5
Female wasp specimens, each identified by “bwsp” followed by
the identification numbers listed below.
505
532
130, 132, 498, 528, 538
122,
113,
139, 475, 516
123, 124, 125, 469, 471, 507
Morph6
Morph7
Morph8
543, 545;
533
542
492
159,119, 480, 526,
G
C
B
M
D
G
495, 522
531, 539
531, 539,
133
121, 491, 497,499,
153, 483,
103, 111, 118, 151, 152, 470, 478, 481, 484, 488, 495, 511, 522
M
A
A
B
K
L
M
Morph
group
Morph1
Morph2
Morph3
Morph4
Morph9
Morph9a
Morph9b
Morph9c
28
F
E1
E2
B
M
L
G
table S6: Summary of data used to delineate species of Bellopius. AFLP clusters with
>50% support (fig. S2) were used to initially define MOTUs, with single gene sequences
and morphological data evaluated independently and used here as supporting elements.
Wasp MOTUs were evaluated at mtCOI based on >1% sequence divergence (fig. S3,
table S3). Reciprocal monophyly at nuclear loci (figs. S4, S5) was considered as
secondary support for MOTUs. Bellopius spH, spI and spJ were all singletons. * = a
single individual showed < 1% divergence (0.8%) between E1 and E2 groups at mtDNA
COI. **= C was monophyletic in MP trees but not reciprocally monophyletic with
respect to D (figs. S4, S5).
AFLP
A
B
C
D
E1
E2
F
G
No data
I
No data
K
L
M
COI
A
B
C
D
E1/E2*
F
G
H
I
J
L/K/M
EF1α
A
B
28S
A
B
C/D
C/D**
E1/G
E2
No data
E1/G
No data
I
No data
E1/G
E2
F
E1/G
No data
I
No data
L/K
L/K
M
M
29
Morphology
Morph9b,9c
Morph4,6,9c
Morph5
Morph8
Morph2
Morph3
Morph1
Morph5,8
No data
No data
No data
Morph9c
Morph4,9c
Morph4,7,9a,9c
Final species
name
spA
spB
spC
spD
spE1
spE2
spF
spG
spH
spI
spJ
spK
spL
spM
table S7: Numbers of puparia obtained from individual flowers (m= male; f= female) of
Gurania acuminata (GA), G. spinulosa (GS)
zero p
1p
2p
3p
4p
5p
fGA
31
9
5
1
1
0
fGS
219
71
13
2
2
1
30
mGA
943
616
17
0
1
0
mGS
1101
545
41
13
4
0
table S8: Counts of Bellopius species in samples of pre- and post-emergence wasps
(excluding singletons, which are spurious “specialists” because they are represented by
just a single specimen) reveal different patterns of host-associations (Fisher Exact Test,
p=0.0075)
Pre-emergence
(wasps in puparia)
Post-emergence
(adult wasps)
Number of Bellopius species with
only one host-fly species
3
10
Number of Bellopius species with
more than one host-fly species
8
1
31
table S9: Number of branches with one or more different Blepharoneura spp (i.e.,
potential for choice by wasps). Categories: singleton branch (i.e., only one single flower
on the branch yielded a fly puparium), and one up to six species of flies on a single
branch. Dead/decayed pupae and branches without flies are excluded.
#species of
flies/branch
fGA
fGS
mGA
mGS
Singleton branch
1
2
3
4
5
6
0
1
1
1
0
0
0
6
2
3
3
0
0
0
30
26
44
21
5
4
0
10
6
18
14
14
8
3
Total # branches with
> 1 infested flower
N=3
N=14
N=130
N=73
Average #spp per
branch
N(all but singleton)
2
2.125
2.17
3.14
N=3
N=8
N=100
N=63
32
table S10: Patterns of “mistakes” by Bellopius parasitoids. This sample includes all
branches bearing flowers in which a Bellopius parasitoid made a “mistake” by
ovipositing into a “lethal” species of Blepharoneura (i.e., a fly species from which an
adult of that species of Bellopius never emerged). Branches were evaluated for the
presence of a “correct” host (a host from which an adult Bellopius of that same species
emerged as an adult). Only one of 16 mistakes occurred on a branch with flowers not
containing the correct host (Fisher’s exact test p= 0.0155). The only “mistake” was made
by a fly belonging to poorly defined E1c lineage (fig. S7).
Plant species
Flower
sex
G. acuminata
G. acuminata
G. acuminata
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
G. spinulosa
male
male
male
female
female
female
male
male
male
male
male
male
male
male
male
male
Branch Bellopius
ID
specimen and
species
34
Bpup24-M
187
Bpup416-E1
204
Bpup323-M
4A
Bpup444-C
4B+C Bpup442-C
4B+C Bpup448-C
29
Bpup289-G
21
Bpup265-K
29
Bpup276-E1
29
Bpup264-G
57
Bpup300-D
180
Bpup180-K
299
Bpup283-B
234
Bpup51-L
11
Bpup286-G
11
Bpu288-G
* see fig. S7 and discussion of spE1
33
Lethal fly
(= mistake)
Correct host
present
Sp30
Sp3
Sp2
sp11
sp11
sp30
Nsp1
Sp30
Sp8
Nsp1
Sp8
Sp30
Sp4
Nsp1
Nsp1
Nsp1
yes
yes
yes
yes
yes
yes
yes
yes
No*
yes
yes
yes
yes
yes
yes
yes