Supplementary Material
Interspecific aggression and character displacement of competitor recognition in
Hetaerina damselflies
Christopher N. Anderson and Gregory F. Grether
Supplemental Experimental Procedures
We generated a phylogeny with H. americana (n=10) and H. occisa (n=8)
individuals from our study sites to assess whether our sampling design captured
independent instances of sympatry with H. titia for both species. Specimens from all sites
were preserved in 95% ethanol in the field and placed in -80º C storage prior to analysis.
In addition to H. americana and H. occisa, we obtained a sequence from Calopteryx
maculate (GenBank accession number: AJ459198.1, Dumont 2005) to serve as an
outgroup. DNA was extracted from thorax muscle tissue using the QIAGEN DNA
Miniprep extraction kit. A 467 bp fragment containing the ITS1, 5.8S and ITS2 region
was amplified and sequenced using published primers (Dumont et al. 2005). Sequences
were aligned using ClustalX and manually optimized (Higgins et al. 1996). No sequence
polymorphisms were observed. The highly conserved 5.8S gene region was used to
position the variable ITS1 and ITS2 regions.
Phylogenetic relationships among the 19 genotypes were estimated using
neighbor joining (NJ), maximum parsimony (equally weighted) (MP), and maximum
likelihood (ML) methods. NJ and MP bootstrap consensus trees were generated from 500
replicates using default settings in MEGA4 (Tamura et al. 2007). The MP tree was
obtained using the close-neighbor-interchange algorithm in which the initial trees were
obtained with the random addition of sequences (10 replicates). For all approaches, all
positions containing gaps/missing data were eliminated from the dataset (complete
deletion option). ML analyses used the model of sequence evolution (TVM+G) selected
by AIC in Modeltest 3.7. Using parameters estimated on a neighbor-joining tree (Saitou
& Nei 1987), we used PAUP* v 4.0b10 (Rogers & Swofford 1998) to perform heuristic
searches [tree bisection–reconnection (TBR) branch swapping; 10 random-addition
replicates] under the maximum-likelihood (ML) optimality criterion. To test the
hypothesis that our sampling design captured independent instances of sympatry, we
generated constrained trees that forced monophyly among sympatric sites within species.
We used PAUP* to conduct Shimodaira–Hasegawa tests (SH) (Shimodaira & Hasegawa
1999) with full optimization and 1000 bootstrap replicates. SH tests assessed whether the
likelihoods of the unconstrained and constrained trees were significantly different.
Phylogenetic relationships among study populations
The gene trees (Figures S1-S3) of H. americana and H. occisa individuals
indicate that phylogenetic relationships are largely interpretable in terms of geography.
For example, H. americana individuals originating from Texas (sites RV, CV, LP) form a
monophyletic group. All topologies indicate that sympatry with H. titia has arisen
independently in both the H. americana and H. occisa clades. ML trees in which
sympatric sites were constrained to be monophyletic were significantly worse compared
to the optimal (unconstrained) trees (H. americana, SH test p<0.001, H. occisa, SH test,
p=0.05).
Figure S1. Neighbor-joining estimate of the phylogenetic relationships of study
populations based on ribosomal gene and spacer data. Bootstrap support, calculated from
500 replicates, is expressed as percentage. Branches corresponding to partitions
reproduced in less than 50% bootstrap replicates are collapsed. Only bootstrap values ≥70
are shown. The scale bar represents 0.05 substitutions/site. Terminal branches are labeled
with the study location, the species, and the sympatry/allopatry status of the site. ALLO:
H. titia absent at the site. SYM: H. titia present at the site. See Figure 1 for geographic
distribution of study locations.
Figure S2. Maximum parsimony estimate of the phylogenetic relationships of study
populations based on ribosomal gene and spacer data. The MP estimate generated 120
most parsimonious trees. The bootstrap consensus tree was inferred from 500 replicates.
Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are
collapsed. The tree is drawn to scale, with branch lengths calculated using the average
pathway method (Nei and Kumar 2000) and are in the units of the number of changes
over the whole sequence. Terminal branches are labeled with the study location, the
species, and the sympatry/allopatry status of the site. ALLO: H. titia absent at the site.
SYM: H. titia present at the site. See Figure 1 for geographic distribution of study
locations.
Figure S3. Maximum likelihood estimate of the phylogenetic relationships of study
populations based on ribosomal gene and spacer data. Bootstrap support, calculated from
200 replicates, is expressed as percentage. Branches corresponding to partitions
reproduced in less than 50% bootstrap replicates are collapsed. The scale bar represents
0.01 substitutions/site. Terminal branches are labeled with the study location, the species,
and the sympatry/allopatry status of the site. ALLO: H. titia absent at the site. SYM: H.
titia present at the site. See Figure 1 for geographic distribution of study locations.
Supplemental Literature Cited
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Phylogenetic relationships, divergence time estimation, and global biogeographic
patterns of calopterygoid damselflies (Odonata, Zygoptera) inferred from
ribosomal DNA sequences. Systematic Biology 54, 347-362.
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Higgins, D. G., Thompson, J. D. & Gibson, T. J. 1996 Using CLUSTAL for multiple
sequence alignments. Computer Methods For Macromolecular Sequence Analysis
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Rogers, J. S. & Swofford, D. L. 1998 A fast method for approximating maximum
likelihoods of phylogenetic trees from nucleotide sequences. Systematic Biology
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Saitou, N. & Nei, M. 1987 The neighbor-joining method - a new method for
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