The Phenology of the Symbiotic Association between Ambystoma maculatum and Unicellular Algae (Oophila) Using Molecular Techniques
The symbiosis between developing Spotted Salamanders (Ambystoma maculatum) and the unicellular alga known as Oophila amblystomatis appears to be mutualistic, with involved parties trading apparent benefits within the salamander egg capsule, but the spatiotemporal ecology of the interaction has yet to be thoroughly explored. Using newly developed primers for quantitative polymerase chain reaction (PCR), we sampled 149 salamander egg masses of various ages from six breeding ponds in northeast Alabama. We documented the presence of algal DNA within egg capsules throughout the developmental process. Regression via linear mixed model estimation showed a positive relationship between the age of egg masses and the probability of algal DNA detection and a negative trend when comparing egg-mass age and qPCR cycle quantification value. These trends indicate an increase in both the probability that a given egg will contain algal DNA and the amount of algal DNA an egg contains, if present, as age increases. We found no effects of pond, site within pond, or year of sampling in either case.ABSTRACT
Nested data structure of Ambystoma maculatum egg-mass sampling locations for this study. Pond HFP was divided into four roughly equal sites by area. Ponds F, G, HW, and M represented one site each. Ponds HFP and F were sampled repeatedly from 2016 to 2019.
The number of Ambystoma maculatum egg-mass samples tested for algae via 18S qPCR detection at each developmental stage and the proportion of those samples that tested positive.
Linear mixed model (LMM) and generalized linear model (GLM) describing the relationships between Ambystoma maculatum egg-mass developmental stage and both quantitative polymerase chain reaction (qPCR) signal strength and probability of containing algal DNA. (A) Random intercept LMM comparing cycle quantification (Cq) value (positive samples only) vs. age of A. maculatum egg mass with random effects of pond, site within pond, and year of sampling included. (B) GLM comparing algae presence vs. developmental stage of A. maculatum egg masses. Bands represent 95% confidence interval. A small amount of random noise was added to differentiate overlapping points.
Phylogenetic and haplotype analyses of algal 18S sequences from various amphibian hosts and locations. (A) Maximum-likelihood tree of algal 18S sequences isolated from Ambystoma maculatum, A. gracile, Lithobates sylvatica, Hynobius negrescens, and L. aurora egg masses collected from eight locations, inferred through 100 parsimony trees and under GTR+gamma+I model. Data matrix included 97 sequences. Bootstrap values based on 1,000 replicates. Bootstrap values above 50 shown at nodes. Black bars on rails to the right of “Oophila” clade indicate amphibian species of egg-mass origin. (B) A minimum spanning haplotype network generated from Oophila sp. sequences of the above matrix (A). Circles represent observed haplotypes from sequence data. Ticks on connecting lines represent the number of presumed mutations separating connected haplotypes.
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