Abstract
BackgroundGammarus minus, a freshwater amphipod living in the cave and surface streams in the eastern USA, is a premier candidate for studying the evolution of troglomorphic traits such as pigmentation loss, elongated appendages, and reduced eyes. In G. minus, multiple pairs of genetically related, physically proximate cave and surface populations exist which exhibit a high degree of intraspecific morphological divergence. The morphology, ecology, and genetic structure of these sister populations are well characterized, yet the genetic basis of their morphological divergence remains unknown.ResultsWe used degenerate PCR primers designed to amplify opsin genes within the subphylum Crustacea and discovered two distinct opsin paralogs (average inter-paralog protein divergence ≈ 20%) in the genome of three independently derived pairs of G. minus cave and surface populations. Both opsin paralogs were found to be related to other crustacean middle wavelength sensitive opsins. Low levels of nucleotide sequence variation (< 1% within populations) were detected in both opsin genes, regardless of habitat, and dN/dS ratios did not indicate a relaxation of functional constraint in the cave populations with reduced or absent eyes. Maximum likelihood analyses using codon-based models also did not detect a relaxation of functional constraint in the cave lineages. We quantified expression level of both opsin genes and found that the expression of both paralogs was significantly reduced in all three cave populations relative to their sister surface populations.ConclusionsThe concordantly lowered expression level of both opsin genes in cave populations of G. minus compared to sister surface populations, combined with evidence for persistent purifying selection in the cave populations, is consistent with an unspecified pleiotropic function of opsin proteins. Our results indicate that phototransduction proteins such as opsins may have retained their function in cave-adapted organisms because they may play a pleiotropic role in other important processes that are unrelated to vision.
Highlights
Gammarus minus, a freshwater amphipod living in the cave and surface streams in the eastern USA, is a premier candidate for studying the evolution of troglomorphic traits such as pigmentation loss, elongated appendages, and reduced eyes
Characterization of G. minus opsin proteins Sequencing of multiple clones of PCR products amplified with the degenerate LWF1A and Scylla opsin primers revealed that there are at least two divergent opsin paralogs, Ops1 and Ops2, in the G. minus genome
Maximum likelihood analysis of the five unique Ops1 and six unique Ops2 amino acid haplotypes, along with various arthropod opsins revealed that both G. minus opsin paralogs clustered within the crustacean long wavelength sensitive (LWS) opsins (Figure 2), bootstrap support for the monophyly of the crustacean LWS clade was moderate (75%)
Summary
A freshwater amphipod living in the cave and surface streams in the eastern USA, is a premier candidate for studying the evolution of troglomorphic traits such as pigmentation loss, elongated appendages, and reduced eyes. A large number of higher taxa contain species restricted to subterranean habitats, and most of these species show convergent differentiation in a suite of features related to the absence of light [1,2]. These features, termed troglomorphy by Christiansen 1962 [3], include partial to complete loss of eyes and body pigment, elaborated extra-optic sensory structures, elongated appendages, and more slender body forms compared to related surface-dwelling taxa. The study of the evolution of troglomorphy in subterranean species is interesting because both elaborated and reduced features are exhibited by the same organism, have evolved independently and repeatedly across many taxa, and are clearly related to the consequences of a single overriding environmental constraint: total darkness
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