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Supporting Information
Methods
Material collected
A total of 31 spittlebug species were used in this study. Taxonomic sampling
covered the major lineages of the spittlebug superfamily (Cercopoidea) based on Cryan
and Svenson (Cryan and Svenson, 2010). Species and collection data are shown in Table
S1. Species identifications were determined by J. Cryan based on morphology, and were
corroborated with DNA sequencing of the insect nuclear locus, 28S rRNA (Cryan et al.,
2000; Cryan and Svenson, 2010). We also included several other species from
throughout Auchenorrhyncha to determine phylogenetic relationships of endosymbiont
lineages, as indicated in Table S1.
Endosymbiont identifications and sequencing
Whole genomic DNA was extracted with a SDS-phenol method. Insect tissues
were homogenized in STE-SDS (0.1 M NaCl, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1%
sodium dodecyl sulfate) and extracted with PCI (phenol: chloroform: isoamyl alcohol =
25:24:1 [v/v]). The aqueous phase was mixed with 1/10 vol of 3 M Sodium acetate (pH
5.2) and 2.5 vol of absolute ethanol, then the mixture was subjected to centrifugation at
15,000 rpm for 5 min to precipitate whole nucleic acid. The pellet was suspended in 400
µL of 10 mM Tris-HCl (pH 8.0).
In order to identify endosymbiont associations, specimens were initially
screened with PCR for Sulcia, Zinderia and Sodalis-like symbionts using symbiontspecific diagnostic primers. Diagnostic PCRs were performed in a reaction mix,
containing 1 x PCR buffer (New England BioLabs), 250 µM dNTPs (5 Prime), 250 nM
each primer (Integrated DNA Technologies), and 0.025 U/µl Taq polymerase (New
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England BioLabs). Thermo-cycling conditions included an initial denaturation step for
95˚C for 2 min; 35x cycle regime of 94˚C for 20 sec, optimized annealing temperatures
for 20 sec (see Table S2), and 72˚C for 1 min; and, a final 72˚C extension phase for 10
min. For taxa that we were unable to amplify Zinderia from, or for those diagnosed to
have the Sodalis-like symbiont, we used PCR cloning and sequencing of partial 16S rRNA
locus with general eubacterial and Sodalis-specific primers (16S1Ab +16SB1, 10F +
1507R, and 16SA1 + Sod1248R: See Table S2) to identify other possible symbionts.
Cloning and sequencing revealed two copies of the 16S rRNA loci in the Sodalis-like
endosymbiont of several species, which were both included in alignments and
downstream phylogenetic analyses. To confirm the presence of one or two bacterial
endosymbiont lineages, based on the presence of two 16S copies, seven taxa
(Aphrophora quadrinotata, Neophilaenus lineatus, Mesoptyelus fasciatus, Philaenarcys
bilineata, Philaenus maghresignus, P. tesselatus, and P. spumarius), were further
subjected to PCR cloning and sequencing of the groEL locus with the SodGroE178F +
SodGroEL1569R primer set (Table S2) PCR amplifications for cloning were performed
using methods for diagnostic PCR as described above. PCR products were ligated with
pGEM-T cloning vector (Promega) and transformed into MAX Efficiency DH5α
Competent Cells (Invitrogen), according to manufacture’s protocol. Colony PCR for
direct sequencing was performed as mentioned above, and PCR amplifications were
treated with 0.05 U/µL Exonuclease I – 0.05 U/µL calf intestine alkali phosphatase (New
England BioLabs) digestion reaction for two 15 min incubation periods at 37˚C and 85˚C.
Cleaned PCR products were sequenced in forward and reverse directions at the Yale
University DNA Analysis Facility.
16S rRNA locus of Sulcia was directly sequenced using 10_CFB_FF + 1515_R
primers (Table S2). Due to high levels of sequence divergence in Zinderia, several
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primer combinations were used for amplification and sequencing of the 16S rRNA locus
(Table S2). PCR products for direct sequencing were amplified and purified as
mentioned above. Finally, 16S rRNA screening and sequencing revealed the presence of
Wolbachia, Arsenophonus, and Rickettsia in some species (Table S3), which are known
facultative symbionts in other insect groups.
Taxon selection, sequence alignment and recombination tests
Sequenced contigs were aligned and edited in Geneious v5.1 (Drummond et al.,
2010). Blastn searches (Altschul et al., 1997) were conducted in GenBank to verify
sequence identity, and to select suitable outgroups to root phylogenies. Related
environmental bacterial and endosymbiont lineages were retrieved in order to
reconstruct global phylogenetic relationships of Sulcia (Bacteriodetes), Zinderia
(betaproteobacteria), and Sodalis-like (gammaproteobacteria) symbionts. For Zinderia,
two separate analyses were run to (a) examine the global relationships of Zinderia
within the betaproteobacteria class, and (b) to reconstruction the relationships and
cophylogenetic correspondence of Zinderia only, with spittlebug hosts. For the global
alignments, sampling aimed to include one representative of each per major
Cercopoidea family. Additionally, species of Betaproteobacteria were chosen to include
lineages determined to be closest relatives of Zinderia, based on blastn searches, as well
as a selection of more distantly related betaproteobacterial species. The sampling for
the Zinderia-only tree included more finely sampled lineages and clade diversity
throughout the Cercopoidea superfamily (e.g., tribal and generic level diversity).
All sequence data was aligned with Muscle v3.5 (Edgar, 2004). Alignments were
subsequently checked by eye for potential algorithmic errors. Sequence alignments for
each symbiont contained highly variable loop regions for which homology was
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ambiguous, and were removed from for phylogenetic analyses: Sulcia, 43-59, & 846855; Zinderia, 108-124, 770-758, 957-969, & 1053-1070; Sodalis-like endosymbiont,
114-123, 523-532, 540-547, 944-928, & 1133-1143. Trimmed data for the global
betaproteobacteria-Zinderia matrix removed large sections of alignable sequence data
for Zinderia lineages, which was recovered for the Zinderia only alignment. For Sulcia
Sodalis-like symbionts and betaproteobacteria phylogenetic analyses, Flavobacterium
columnare, Pseudomonas aeruginosa and Neisseria gonorrhoeae were used as an
outgroup, respectively, since these bacterial species are known to be positioned basally
in each bacterial groups. A suitable outgroup for Zinderia only phylogenetic analyses
was selected based on the results from the global betaproteobacterial phylogenetic
analyses.
Untrimmed Sodalis alignments were examined for potential recombination
between endosymbiont lineages and between the dual 16S rRNA copies. Previous work
suggests that inference of recombination can vary between methods (Wiuf et al., 2001);
thus, we implemented RDP4 program package (Martin et al., 2005) and the likelihood
based GARD method (Kosakovsky Pond et al., 2006). Both GARD and RDP4 are able to
detect potential breakpoints in an alignment; however, RDP4 allows for identification of
possible recombinant sequences. GARD was run on the DATAMONKEY server (Delport
et al., 2010) under the HKY85 nucleotide substitution model determined by the Akaike
Information Criterion (AIC). Likelihood tests for recombination screened the entire
alignment for potential breakpoints and estimated the AICc to select the most likely
scenario. Statistical significance was assessed with the Kishino-Hasegawa test (KH),
which examines phylogenetic incongruence between recombinant partitions of the
alignment.
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RDP4 was run with all seven available models (RDP, Martin et al., 2005; SiScan,
Gibbs et al., 2000; Bootscan, Salminen et al., 1995; 3SEQ,Boni et al., 2007; Chimaera,
Posada and Crandall, 2001; MaxChi, Maynard Smith, 1992; and, GENECONV, Padidam et
al., 1999), and positive results were rechecked with LARD (Holmes et al., 1999) and
PhylPro (Weiller, 1998). Recombination events were determined to be real if they met
the following criteria: (i) two or more programs corroborated the same event at similar
breakpoints, (ii) events are statistically significant, with any reported as “trace evidence”
discarded, (iii) the recombinant has a consensus score of >0.40 (authors suggest that a
score between 0.40-0.50 indicates a possible, but unlikely error, and a score >.60
recombination is certain), and iv) breakpoint plots are consistent with the MaxChi
coordinates.
Phylogenetic analyses
Phylogenetic relationships of all three endosymbionts were inferred with
Maximum Likelihood (ML) and Bayesian methods. Likelihood models of nucleotide
substitution were estimated for each alignment using JModeltest v.2 and selected with
the Bayesian Information Criterion (Darriba et al., 2012). ML runs were conducted with
RAxML v7.3.2 (Stamatakis, 2006) on CIPRES (Miller et al., 2009). RAxML was run under
a GTR+GAMMA model of nucleotide evolution with 25 rate categories for both the
inference of the ML tree and for 1000 bootstrap replicates.
Bayesian phylogenetic inference was done using MPI-MrBayes 3.1.2
(Huelsenbeck and Ronquist, 2001) on Xsede cluster in CIPRES (Miller et al., 2009). Two
independent searches for the posterior optima were conducted with four-chains each
for 1.5 – 2.0 x 107 generations sampled every 1000th iteration. Searches were run under
the appropriate nucleotide substitution model for each endosymbiont dataset. Chain
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convergence was assessed throughout the length of the run by monitoring the average
standard deviation of split frequencies (ASDF < 0.05). Burn-in and chain convergence
were determined with the potential scale reduction factor (PSRF = 1.0), and by plotting
run statistics with the cumulative function in AWTY (Nylander et al., 2008) and in
Tracer v1.6 (Rambaut and Drummond, 2009). A 50% majority consensus tree was
assembled from post burn-in iterations.
Co-phylogenetic analysis
Congruence between the spittlebug hosts and Zinderia was statistically assessed
using Jane v.4 (Conow et al., 2010). Jane implements an event cost (e.g., co-speciation,
host-switching, and loss) algorithm that optimizes co-phylogenetic reconstruction by
iteratively searching for solutions that minimize the total number of costs (Conow et al.,
2010). Since optimal event costs are not directly known, Jane was set to run across a
range of values from zero to three. Due to low sequence divergence, phylogenetic
hypotheses for both host and bacterial symbionts resulted in soft polytomies. Thus, Jane
was set to sequentially resolve all polytomies in rapid succession for co-phylogenetic
comparisons while finding the optimal branching solution. Since no phylogenetic
hypothesis exists for the placement of the spittlebug species, Eurylax carnifex, screened
in this study, we removed it from cophylogenetic analyses. Additionally, our taxonomic
sampling replaced Clastoptera obtusa with the congeneric species, Clastoptera
arizonana, which, given their close relationships, should not affect global inference of
codiversification. Statistical significance was assessed by randomly generating a null
distribution of 1000 trees with randomized tip values. Final outputs were additionally
assessed with co-phylogenetic branch support derived from the proportion of iterations
that found the same solutions.
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Fluorescence in situ hybridization
In order to localize and verify endosymbiont associations, whole mount and
paraffin sectioning Fluorescence in situ Hybridization (Fukatsu et al, 1998; Koga et al.,
2009) experiments (FISH) were performed on nymphs of both Philaenus spumarius
(Sodalis) and Clastopetera arizonana (Zinderia). Fluorescing oligonucleotide probes
targeting identified endosymbionts were designed as follows: P. spumarius for Sulcia
(TYE665-Sul664R) and Sodalis-like symbiont (Al555-Sod1248R); and, C. arizonana for
Sulcia (TYE665-Sul664R) and Zinderia (TYE563-Bet940R)(Table S2). Field-collected
specimens were initially preserved in acetone (Fukatsu, 1999). For dissection,
specimens were transferred to 70% ethanol and had their legs removed to facilitate
permeation of reagents into the insect tissues. Dissected material was fixed by
overnight incubation in Carnoy’s solution (ethanol: chloroform: acetic acid = 6: 3: 1
[v/v]) at room temperature (RT). Fixed material was then treated with 6% hydrogen
peroxide in 80% ethanol for one week to quench autofluorescence in insect tissues.
After bleaching, specimens were rinsed with absolute ethanol and stored at -20 oC until
use.
For FISH of tissue sections, paraffin-embedded tissue sections were prepared
and hybridized as previously described (Koga et al., 2009). Briefly, the H2O2-treated
tissues were embedded in paraffin with an ethanol-xylene-paraffin treatment series.
Serial tissue sections of 5 µm in thickness were prepared by using a rotary microtome
(Leica) and mounted on silane-coated glass slides. The sections were dewaxed through
series of xylene, absolute ethanol, and RNase-free water before hybridization. Tissue
sections were hybridized in 150 µL of hybridization buffer (20mM Tris-HCl [pH 8.0],
0.9M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide, 100 pmol/ml each of the
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probes and 1 µg/mL of Hoechst 33342) in a humidified chamber and incubated at RT
overnight. The sections were then briefly washed with PBSTx (0.8% NaCl, 0.02% KCl,
0.115% Na2HPO4, 0.02% KH2PO4, 0.3% Triton X-100), mounted in DABCO-glycerol-PBS
(13.7 mM NaCl, 0.81 mM Na2HPO4, 0.27 mM KCl, 0.15 mM KH2PO4 [pH 7.5], 90% [v/v]
glycerol, 1.25% (w/v) 1,4-Diazabicyclo[2.2.2]octane) and observed under an
epifluorescence microscope (Eclipse TE2000-U; Nikon). Images were acquired with NIS
Elements BR Ver. 3.10. The brightness and contrast were adjusted with PhotoShop
CS5.1 (Adobe). Whole body FISH images of P. spumarius (Fig. 3D) and C. arizonana (Fig.
3I) were generated by merging multiple images using this application.
For whole mount FISH, H2O2-treated specimens were rehydrated in PBSTx, and
hybridized in 500 ul of hybridization buffer containing 100 pmol/mL each of probes
and incubated at RT overnight with gentle agitation. Hybridized specimens were
washed thoroughly with PBSTx, mounted in DABCO-glycerol-PBS, and observed as
described above.
For comparison of bacteriome morphology, adults of the membracid Enchenopa
permutata and deltocephaline leafhopper Deltocepahlus nr.flavicosta were also
subjected to whole-mount FISH with the probes TYE665-Sul664R and TYE563-Bet940R
(Table S2). After hybridization, terga which the bacteriomes were associated with were
detached from the treehopper body, then were mounted and observed as mentioned
above. On the other hand, the leafhopper specimen was whole-mounted in Slow-fade
antifade solution (Invitrogen) and observed under a laser confocal microscope
(LSM710; Zeiss) and with Zen 2011 (Carl-Zeiss).
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