Causes and Consequences of Lung Loss in Salamanders

Abstract

Lungs were once thought to be a universal feature of tetrapods and essential for vertebrate life on land. This view changed in the late 19th century with the discovery of several salamander species that lack lungs. Since these species are descendants of lunged ancestors, the absence of lungs must represent an instance of evolutionary loss. Further study has revealed several independent losses of lungs across amphibians, including at the base of the salamander family Plethodontidae. Plethodontids comprise over two thirds of all living salamander species, yet many features of lungless salamander biology are unknown. My dissertation investigates the evolution and development of lung loss, including the genetic basis for lunglessness and the consequences of lunglessness for respiration and the circulatory system.

I determined that plethodontid salamanders are not entirely lungless; lungs actually begin to form in the embryo. This includes lung specification, the formation of a lung primordium, and conserved expression of genetic markers of lung differentiation. However, the lung primordium subsequently regresses by apoptosis, yielding adults with no trace of a vestigial lung. Transcriptome sequencing of the lung primordia of lunged and lungless salamanders suggests a role for increased Tgf-beta signaling in lung regression. I established that Tgf-beta represses lung development in other species of salamanders, providing support for its role in lung loss.

Plethodontid salamanders perform gas exchange through their skin and lining of the mouth (extrapulmonary respiration). I discovered a novel gene in salamanders that is potentially neofunctionalized for extrapulmonary respiration in plethodontids. The lung-specific gene encoding surfactant-associated protein C (SPC) is duplicated in salamanders. Both paralogs of this gene are expressed in the lung of lunged salamanders, representing the ancestral expression pattern of SPC in tetrapods. In contrast, lungless salamanders express a paralog of SPC in extrapulmonary sites of gas exchange. These sites include the skin during the aquatic larval stage and the lining of the mouth in terrestrial adults. I propose that extrapulmonary expression of this paralog in salamanders reduces the thickness of the mucus layer that covers the respiratory surfaces and aids gas exchange.

The lungs function as part of an integrated cardiopulmonary system. In animals with lungs, the atrial septum helps to separate oxygenated and deoxygenated blood in the heart. I characterized cardiac anatomy within a broad sample of lunged and lungless salamanders. I found that independent lineages of lungless salamanders convergently evolved a reduced and non-functional atrial septum, resulting in blood flow between the two atrial chambers. In mammals, formation of the atrial septum is dependent on morphogens secreted from the developing lungs. I provide evidence that atrial septum reduction in plethodontid salamanders is a direct consequence of loss of these signaling interactions due to lung regression. Developmental interaction between the heart and lungs may mediate the coordinated evolution of the cardiopulmonary system, ensuring that the atrial septum develops in the presence of lungs but does not fully form in lungless species, where it would be disadvantageous.