Journal of Chemical Ecology
Appendix S1: Representation of extrafloral nectar composition presented in the behavioral tests used to determine differential utilization of EFN
Artificial mimics which represented EFN produced by floral buds and young leaves of Humboldtia brunonis at the three sites Agumbe, Sampaji and Solaikolli were made prior to behavioral experiments. In order to choose samples that represented floral bud and young leaf EFN from a particular site, we used a criterion obtained from the results of the behavioral tests performed on the ability of ants to differentially utilize different concentrations of sugars (Fig. 2 of the main text). The results of these behavioral tests revealed that the three dominant ant species at each site were able to differentiate and preferentially utilize sugar (sucrose,glucose or fructose) solutions of concentrations above 30% (w/v). Hence, all samples of floral bud or young leaf EFN from a particular site that varied in the concentration of any one sugar by >25% (w/v) viz.floral bud EFN from Agumbe, young leaf EFN from Sampaji, floral bud and young leaf EFN from Solaikolliwere represented by two EFN-mimicking solutions instead of one. In order to determine the sugar and amino acid concentrations of these EFN-mimicking solutions, we performed a cluster analysis using the concentration of various sugars and amino acids as initial raw data input (Fig. S1) and made EFN mimics using the median value of the sugars and amino acids in each of the two clusters. In order to determine distances between clusters we used the ‘single linkage’ rule which determines distance between clusters based on the distance of the two closest objects (nearest neighbors). Euclidean distances were used to compute distances between objects. All components (sugars and amino acids) that were not detected in a particular sample of EFN were given the value of zero and subsequently deleted case-wise from the analysis. For samples of floral bud or young leaf EFN from a site that varied in the concentration of all sugars by <25% (w/v) viz.young leaf EFN from Agumbe and floral bud EFN from Sampaji only one EFN-mimicking solution was made using the median values of the concentration of sugars and amino acids in all the samples. Concentrations of sugars and amino acids to make the different floral bud and young leaf EFN-mimicking solutions are presented in Table S1.
Figure S1: Cluster analysis of extrafloral nectar samples based on composition. Extrafloral nectar mimics were made using the median concentrations of samples in the high conc. EFN and low conc. EFN clusters. a) Bud EFN from Agumbe; b) Leaf EFN from Sampaji; d) Bud EFN from Solaikolli; d) Leaf EFN from Solaikolli.
Shenoy et al. (2010) Composition of extrafloral nectar influences interactions between the myrmecophyte Humboldtia brunonis and its ant associates: implications for the evolution of myrmecophytism
Journal of Chemical Ecology
Table S1: Composition of solutions that mimic the composition of floral bud EFN and young leaf EFN produced by Humboldtia brunonis at three sites (%w/v)
Floral bud EFN mimics / Young leaf EFN mimicsSite / Agumbe / Agumbe / Sampaji / Solaikolli / Solaikolli / Agumbe / Sampaji / Sampaji / Solaikolli / Solaikolli
Within-site Concentration / low / high / low / high / low / high / low / high
Resource Ratios (Total Sugars/Total Amino Acids) / 78 / 56 / =42.3/0 / =49.6/0 / =88.8/0 / 213 / =36.4/0 / =92.8/0 / =17.4/0 / 44
Sugars/amino acids
Sucrose / 17.1 / 40.3 / 12.9 / 18.4 / 27.6 / 5.0 / 5.6 / 19.4 / 1.9 / 7.8
Glucose / 5.1 / 19.1 / 6.9 / 31.2 / 28.5 / 2.3 / 9.0 / 19.1 / 14.7 / 11.0
Fructose / 12.5 / 34.5 / 16.7 / 0 / 30.6 / 3.9 / 14.6 / 38.3 / 0 / 13.7
Galactose / 4.8 / 13.2 / 5.0 / 0 / 0 / 1.5 / 6.2 / 13.3 / 0.8 / 2.1
Inositol / 0.5 / 1.2 / 0.8 / 0 / 2.1 / 0.1 / 1.0 / 2.7 / 0 / 1.1
Total sugars / 40 / 108.3 / 42.3 / 49.6 / 88.8 / 12.8 / 36.4 / 92.8 / 17.4 / 35.7
Isoleucine (E)* / 0.03 / 0.2 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0.06
Leucine (E) / 0.2 / 0.6 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0.08
Methionine (E) / 0 / 0.08 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
Phenylalanine (E) / 0.2 / 0.9 / 0 / 0 / 0 / 0.06 / 0 / 0 / 0 / 0.3
Threonine (E) / 0.02 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0.01
Tryptophan (E) / 0 / 0.07 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
Valine (E) / 0.02 / 0.07 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0.05
Alanine (N)* / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0.3
Aspartic acid (N) / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0.008
Serine (N) / 0.04 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
Total amino acids / 0.51 / 1.92 / 0 / 0 / 0 / 0.06 / 0 / 0 / 0 / 0.81
(E)*: essential amino acid; (N)*: non-essential amino acid
Table S2: Concentration of the various sugars and amino acids (g/100 ml), that are not shown in Table 1, in EFN of floral bud inflorescences and young leaves of Humboldtia brunonis from three sites [Mean ± SD (Median)]; components followed by different letters in comparisons between floral bud and young leaf EFN, ‘within a site or pooled across sites’, are significantly different (Mann-Whitney U tests andBonferroni corrections)
EFN source / Floral Bud EFN / Young Leaf EFNSITE / Pooled across sites / Agumbe / Sampaji / Solaikolli / Pooled across sites / Agumbe / Sampaji / Solaikolli
N=28 / N=10 / N=7 / N=11 / N=28 / N=11 / N=9 / N=8
Monosaccharides:
Reducing hexoses
Maltose / 0 / 0 / 0 / 0 / 0.001 ± 0.003
(0) / 0 / 0 / 0.002 ± 0.01
(0)
Non-reducing hexoses
Galactose / 5.2 ± 4.8
(4.2) / 9.6 ± 4.7
(11.1) a / 5.0 ± 2.1
(5) / 1.3 ± 1.9
(0.7) / 4.6 ± 6.3
(1.7) / 1.9 ± 1.4
(1.5) b / 10.9 ± 8.1
(11.1) / 1.4 ± 1.0
(1.1)
Mannose / 0.2 ± 1.0
(0) / 0 / 0 / 0.5 ± 1.5
(0) / 0.2 ± 1.2
(0) / 0 / 0.7 ± 2.
(0) / 0.1 ± 0.2
(0)
Altrose / 0.1 ± 0.5
(0) / 0 / 0.4 ± 1.0
(0) / 0 / 0 / 0 / 0 / 0
Reducing pentose
Arabinose / 0.7 ± 2.8
(0) / 0 / 2.7 ± 5.4
(0) / 0 / 0.01 ± 0.1
(0) / 0 / 0.04 ± 0.
(0) / 0
Non-reducing pentoses
Ribose / 1.1 ± 3.9
(0) / 0 / 3.7 ± 7.4
(0) / 0.4 ± 1.2
(0) / 0 / 0 / 0 / 0
Xylose / 0.04 ± 0.2
(0) / 0 / 0 / 0.1 ± 0.3
(0) / 0.3 ± 1.6
(0) / 0 / 1.0 ± 2.9
(0) / 0
Unusual sugars
Inositol / 0.8 ± 1.0
(0.6) / 1.0 ± 0.5
(1.0) a / 1.1 ± 0.8
(0) / 0.6 ± 1.4
(0) / 0.9 ± 1.1
(0.3) / 0.2 ± 0.2
(0.1) b / 1.8 ± 1.2
(0) / 0.6 ± 0.9
(0.2)
Arabinoic acid / 0.01 ± 0.04
(0) / 0 / 0.03 ± 0.1
(0) / 0 / 0 / 0 / 0 / 0
Essential amino acids
Isoleucine / 0.04 ± 0.1
(0) / 0.1 ±0.1
(0.1) a / 0 / 0.01 ± 0.03
(0) / 0.01 ± 0.03 (0) / 0.01 ± 0.01
(0) b / 0 / 0.03 ± 0.05
(0)
Leucine / 0.2 ± 0.3
(0) / 0.4 ± 0.3
(0.5) a / 0 / 0.03 ± 0.09
(0) / 0.03 ± 0.06 (0) / 0.04 ± 0.05
(0) b / 0 / 0.05 ± 0.1
(0)
Methionine / 0.02 ± 0.03
(0) / 0.05 ± 0.04
(0.04) a / 0 / 0 / 0.001 ± 0.004
(0) / 0.003 ± 0.01 (0) b / 0 / 0
Phenylalanine / 0.2 ± 0.4
(0) / 0.6 ± 0.4
(0.7) a / 0 / 0 / 0.1 ± 0.1
(0) / 0.1 ± 0.1 (0.1) b / 0 / 0.2 ± 0.2
0
Threonine / 0.005 ± 0.01 (0) / 0.01 ± 0.02
(0) / 0 / 0 / 0.002 ± 0.01
(0) / 0 / 0 / 0.01 ± 0.01
(0)
Tryptophan / 0.03 ± 0.1
(0) / 0.1 ± 0.2
(0.008) a / 0 / 0 / 0.001 ± 0.003
(0) / 0.001 ± 0
(0) b / 0 / 0
Valine / 0.04 ± 0.1
(0) / 0.1 ± 0.2
(0.02) / 0 / 0.02 ± 0.06
(0) / 0.01 ± 0.02
(0) / 0.003 ± 0.01 (0) / 0 / 0.02 ± 0.04
(0)
Non-essential amino acids
Alanine / 0 / 0 / 0 / 0 / 0.05 ± 0.1
(0) / 0.01 ± 0.02 (0) / 0 / 0.1 ± 0.2
(0)
Aspartic acid / 0.001 ± 0.01
(0) / 0 / 0.006 ± 0.01
(0) / 0 / 0.01 ± 0.05
(0) / 0 / 0.03 ± 0.1
(0) / 0.005 ± 0.2
(0)
Glutamine / 1.1 ± 3.5
(0) / 2.6 ± 5.5
(0) / 0 / 0.4 ± 1.4
(0) / 0.07 ± 0.2
(0) / 0.07 ± 0.2 (0) / 0 / 0.1 ± 0.4
(0)
Glycine / 0.01 ± 0.03
(0) / 0.02 ± 0.05
(0) / 0 / 0 / 0.003 ±0.01
(0) / 0.008 ± 0.02 (0) / 0 / 0
Proline / 0.01 ± 0.05
(0) / 0.03 ± 0.1
(0) / 0 / 0 / 0 / 0 / 0 / 0
Serine / 0.04 ± 0.1
(0) / 0.1 ± 0.2
(0) / 0 / 0 / 0.003 ± 0.01
(0) / 0.002 ± 0.01 (0) / 0 / 0.01 ± 0.02
(0)
Tyrosine / 0 / 0 / 0 / 0 / 0.001 ± 0.01
(0) / 0 / 0 / 0.004 ± 0.01
(0)
Unidentified amino acids
Unidentified 1 / 0.2 ± 0.4
(0) / 0.5 ± 0.5
(0.5) a / 0.04 ± 0.1
(0) / 0.03 ± 0.1
(0) / 0.2 ± 0.4
(0) / 0.1 ± 0.1 (0.05) b / 0.2 ± 0.6
(0) / 0.2 ± 0.3
(0)
Unidentified 2 / 0.0005 ± 0.003
(0) / 0 / 0.002 ± 0.005
(0) / 0 / 0 / 0 / 0 / 0
Shenoy et al. (2010) Composition of extrafloral nectar influences interactions between the myrmecophyte Humboldtia brunonis and its ant associates: implications for the evolution of myrmecophytism