Research Article

Passive accumulation of alkaloids in inconspicuously colored frogs refines the evolutionary paradigm of acquired chemical defenses

Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, United States
Department of Integrative Biology and Biodiversity Collections, University of Texas at Austin, United States
Smithsonian Tropical Research Institute, Panama
Grupo de Investigación en Ecología Evolutiva en los Trópicos (EETROP), Universidad de las Américas, Ecuador
Ecological Networks Lab, Technische Universität Darmstadt, Germany
Departamento de Ciencias Biológicas, Universidad de los Andes, Colombia
Department of Biology, Stanford University, United States
Max Planck Institute for Evolutionary Biology, Germany
Department of Chemistry and Physics, Indiana State University, United States
Museo de Zoología, Escuela de Biología, Facultad de Ciencias Exactas y Naturales, Pontificia Universidad Católica del Ecuador, Ecuador
Department of Biological Sciences, St John’s University, United States

Dec 27, 2024

https://doi.org/10.7554/eLife.100011.3

Open access
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Figures
Tables
Additional files

3 figures, 1 table and 7 additional files

Figures

Figure 1

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A new evolutionary model of toxin sequestration in Dendrobatidae.

We propose that alkaloid consumption, some level of alkaloid resistance, and passive accumulation were present in the most recent common ancestor of Dendrobatidae; enhanced resistance and sequestration mechanisms then arose later, resulting in the chemical defense phenotype. Our model places less emphasis on dietary changes compared to prior studies, and more strongly emphasizes novel molecular mechanisms (e.g. binding proteins and target-site insensitivity; Alvarez-Buylla et al., 2023; Tarvin et al., 2017; Tarvin et al., 2016). Purple lines indicate lineages with chemical defense. Gray lines indicate lineages that putatively lack chemical defense. All images of frogs were taken by RDT and are identified by their museum number.

Figure 2

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From left to right: an ultrametric tree showing phylogenetic relationships inferred previously (Wan et al., 2023) among sampled species with the three defended poison frog clades highlighted in purple, the undefended clades in dark gray, and non-dendrobatids in light gray (Bufonidae: *Amazophrynella siona* and *Atelopus* aff. *spurrelli*; Leptodactylidae: *Lithodytes lineatus*).

Tile color indicates the log of the total quantity of alkaloids in each class as measured by the sum of integrated areas of alkaloids of that class from GC-MS data per individual. The number in each tile indicates the number of alkaloids (including isomers) detected in each individual for each class. On the right are prey items recovered from the stomach of each individual, colored by arthropod group and scaled to 1 (total number of prey identified are shown under N). Note the large proportion of ants (Formicidae, dark purple) and mites (Acari, light purple) in many of the individuals compared to other prey types. See Supplementary file 1 for raw diet data and Supplementary file 4 for full alkaloid data. Poison-frog genera names are abbreviated as follows: *All*., *Allobates; Ame., Ameerega; And*., *Andinobates; D*., *Dendrobates; E*., *Epipedobates; H*., *Hyloxalus; Le*., *Leucostethus; O*., *Oophaga; P., Phyllobates; R*., *Rheobates; S*., *Silverstoneia*; Alkaloid class abbreviations are based on Daly et al., 2009; Daly et al., 2005 and are as follows: HTX, histrionicotoxins; PTX, pumiliotoxins; PTXB, pumiliotoxin B; aPTX, allopumiliotoxins; DeoxyPTX, deoxypumiliotoxins; hPTX, homopumiliotoxins; deoxy-hPTX, deoxy-homopumiliotoxins; DHQ, decahydroquinolines; NMeDHQ, N-methyldecahydroquinolines; HO-DHQ, hydroxy-decahydroquinolines; 3,5 P, 3,5-disubstituted pyrrolizidines; HO-3,5-P, hydroxy-3,5-disubstituted pyrrolizidines; 5-I, 5-substituted indolizidines; 3,5-I, 3,5-disubstituted indolizidines; 5,6-I, 5,6-disubstituted indolizidines; 5,8-I, 5,8-disubstituted indolizidines; Dehydro-5,8-I, dehydro-5,8-indolizidines; 5,6,8-I, 5,6,8-trisubstituted indolizidines; HO-5,6,8-I, hydroxy-5,6,8-trisubstituted indolizidines; 1,4-Q, 1,4-disubstituted quinolizidines; 4,6-Q, 4,6-disubstituted quinolizidines; 3,5-Q, 3,5-disubstituted quinolizidines; 1,3,4-Q, 1,3,4-trisubstituted quinolizidines; Lehm, lehmizidines; Epiquinamide, epiquinamide; 2-Pyr, 2-substituted pyrrolidine; 3-Pyr, 3-substituted pyrrolidine; 2,5-Pyr, 2,5-disubstituted pyrrolidines; Pyr, pyrrolizidine of indeterminate substitution; 2,6-Pip, 2,6-disubstituted piperidines; Pip, other piperidines; Pyri, pyridines (including epibatidine); GTX, gephyrotoxins; Tricyclic, coccinelline-like tricyclics; SpiroP, spiropyrrolizidines; Necine, unspecified necine base; Unclass, unclassified alkaloids without known structures.

Figure 3

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Hypothesized physiological processes that interact to determine the defense phenotype: toxin intake, toxin elimination (Elim.), and toxin sequestration (Seq.).

A new paradigm: the passive-accumulation hypothesis for definitions. Although toxin intake sets a maximum for the total possible amount of toxin accumulation (Acc.), it cannot fully explain the defensive phenotype. We hypothesize that an undefended “no accumulation” phenotype is characterized by the absence of any ability to sequester toxins in combination with a high rate of elimination, resulting in 0 toxin accumulation (dashed gray lines); this phenotype is a likely ancestral state for many animals. In contrast, we hypothesize that an undefended passive-accumulation phenotype is characterized by lower elimination than the no accumulation phenotype, leading to a low amount of toxin accumulation (yellow lines). We hypothesize that a defended sequestration phenotype evolves from an intermediate passive-accumulation phenotype through the addition of novel sequestration mechanisms, and possibly even lower elimination rates, that result in high toxin accumulation and the defended phenotype (purple lines).

Tables

Table 1

Range and median of alkaloid quantity (estimated by the sum of integrated areas) and alkaloid diversity (number of different compounds) by species from the GC-MS assessment.

The presumed chemical defense phenotype for poison frogs is given according to Santos and Cannatella, 2011. Purple rows highlight defended species. *From a UHPLC-HESI-MS/MS dataset for which alkaloids were not quantified. Note that the UHPLC-HESI-MS/MS and GC-MS assays differed in both instrument and analytical pipeline, so ‘Alkaloid Number’ values from the two assay types should not be compared to each other directly.

Family	Subfamily	Species	Phenotype	Sample Size (frogs)	Log (Total Integrated Area)		Alkaloid Number
Family	Subfamily	Species	Phenotype	Sample Size (frogs)	Range	Median	Range	Median
Dendrobatidae	Aromobatinae	Rheobates palmatus	undefended	4	13.07–14.24	14.00	1–4	1.5
Dendrobatidae	Aromobatinae	Allobates insperatus	undefended	8	13.47–15.44	14.99	1–9	5.0
Dendrobatidae	Aromobatinae	Allobates juanii	undefended	1	14.10	14.10	1	1.0
Dendrobatidae	Aromobatinae	Allobates kingsburyi	undefended	1	13.63	13.63	2	2.0
Dendrobatidae	Aromobatinae	Allobates talamancae	undefended	3	14.89–16.27	15.09	2–4	3.0
Dendrobatidae	Aromobatinae	Allobates zaparo	undefended	1	16.78	16.78	8	8.0
Dendrobatidae	Colostethinae	Leucostethus fugax	undefended	8	12.57–15.33	14.00	3–8	4.5
Dendrobatidae	Colostethinae	Ameerega bilinguis	defended	1	21.97	21.97	133	133.0
Dendrobatidae	Colostethinae	Ameerega hahneli	defended	4	20.21–22.29	21.68	85–140	128.5
Dendrobatidae	Colostethinae	Silverstoneia flotator*	undefended	12	NA	NA	0–1	0.0
Dendrobatidae	Colostethinae	Silverstoneia aff. gutturalis	undefended	9	11.80–17.33	15.40	1–10	3.0
Dendrobatidae	Colostethinae	Silverstoneia erasmios	undefended	2	14.70–16.11	15.41	15–15	15.0
Dendrobatidae	Colostethinae	Epipedobates aff. espinosai	defended	2	18.44–20.20	19.32	83–131	107.0
Dendrobatidae	Colostethinae	Epipedobates anthonyi	defended	1	20.54	20.54	127	127.0
Dendrobatidae	Colostethinae	Epipedobates boulengeri	defended	2	18.87–19.39	19.13	77–94	85.5
Dendrobatidae	Colostethinae	Epipedobates currulao	defended	2	19.49–19.68	19.59	99–105	102.5
Dendrobatidae	Colostethinae	Epipedobates espinosai	defended	2	18.82–21.33	20.08	85–146	115.5
Dendrobatidae	Colostethinae	Epipedobates machalilla	defended	2	12.98–15.67	14.32	8–38	23.0
Dendrobatidae	Colostethinae	Epipedobates tricolor	defended	2	18.36–19.07	18.72	91–114	102.5
Dendrobatidae	Hyloxalinae	Hyloxalus awa	undefended	7	0.00–16.05	13.58	0–12	3.0
Dendrobatidae	Hyloxalinae	Hyloxalus shuar	undefended	1	14.92	14.92	5	5.0
Dendrobatidae	Hyloxalinae	Hyloxalus sp. Agua Azul	undefended	1	14.30	14.30	8	8.0
Dendrobatidae	Hyloxalinae	Hyloxalus toachi	undefended	2	0.00–0.00	0.00	0–0	0.0
Dendrobatidae	Dendrobatinae	Phyllobates aurotaenia	defended	4	17.72–21.08	18.88	48–118	67.5
Dendrobatidae	Dendrobatinae	Dendrobates truncatus	defended	3	20.05–23.95	20.42	111–172	115.0
Dendrobatidae	Dendrobatinae	Oophaga sylvatica	defended	5	22.86–24.85	23.76	152–189	175.0
Dendrobatidae	Dendrobatinae	Andinobates fulguritus	defended	2	20.09–20.51	20.30	80–85	82.5
Dendrobatidae	Dendrobatinae	Andinobates minutus	defended	4	16.57–18.77	18.07	34–80	66.0
Bufonidae		Amazophrynella siona	NA	2	14.12–14.40	14.26	1–1	1.0
Bufonidae		Atelopus aff. spurrelli	NA	1	11.58	11.58	4	4.0
Eleutherodactylidae		Eleutherodactylus cystignathoides*	NA	3	NA	NA	0–0	0.0
Leptodactylidae	Leptodactylinae	Lithodytes lineatus	NA	2	0.00–0.00	0.00	0–0	0.0