All life on Earth started out under water. However, around 400 million years ago some vertebrates, such as fish, started developing limbs and other characteristics that allowed them to explore life on land. One of the most pivotal features to evolve was the lungs, which gave vertebrates the ability to breathe above water.

Most land-living vertebrates, including humans, have two lungs which sit on either side of their chest. The lungs extract oxygen from the atmosphere and transfer it to the bloodstream in exchange for carbon dioxide which then gets exhaled out in to the atmosphere. How this important organ first evolved is a hotly debated topic. This is largely because lung tissue does not preserve well in fossils, making it difficult to trace how the lungs of vertebrates changed over the course of evolution.

To overcome this barrier, Cupello et al. compared the lungs of living species which are crucial to understand the early stages of the water-to-land transition. This included four species of lunged bony fish which breathe air at the water surface, and a four-legged salamander that lives on land.

Cupello et al. used a range of techniques to examine how the lungs of the bony fish and salamander changed shape during development. The results suggested that the lungs of vertebrates started out as a single organ, which became truly paired later in evolution once vertebrates started developing limbs. This anatomical shift increased the surface area available for exchanging oxygen and carbon dioxide so that vertebrates could breathe more easily on land.

These findings provide new insights in to how the lung evolved into the paired structure found in most vertebrates alive today. It likely that this transition allowed vertebrates to fully adapt to breathing above water, which may explain why this event only happened once over the course of evolution.

Lungs, the most important organ of the pulmonary complex, are rarely preserved in fossils, hindering direct evidence of how the earliest air-breathing vertebrates breathed air. Indeed, among fossil taxa, the presence of lungs has only been confirmed in coelacanths (Brito et al., 2010; Cupello et al., 2017b; Cupello et al., 2019), a salamander (Tissier et al., 2017), an ornithuromorph bird (Wang et al., 2018), and possibly a tadpole (Rossi et al., 2019). In addition, the presence of specialized skeletal structures, such as cranial ribs and enlarged spiracular openings, provided indirect evidence of air-breathing behaviour in a wider range of fossil sarcopterygians including lungfishes and early tetrapods, but also actinopterygians (Long, 1993; Long et al., 2006; Clement and Long, 2010; Clement et al., 2016; Cupello et al., 2019).

Altogether, the evolutionary origin of the vertebrate lung can therefore be narrowed down to osteichthyans (Goujet, 2011; Tatsumi et al., 2016). The interpretation of thoracic paired masses in the antiarch placoderm Bothriolepis canadensis as a lung (Denison, 1941; Arsenault et al., 2004) even suggested an origin among early jawed vertebrates, dating back to at least the Devonian period. Yet, lung affinities for such structures remain elusive (Janvier et al., 2007) and could not be confirmed by anatomical, phylogenetic, or biological data (Goujet, 2011; Béchard et al., 2014). Here, we follow Janvier et al., 2007, Goujet, 2011 and Béchard et al., 2014, and consider that observable evidences are elusive and do not support the interpretation of these paired masses as a lung.

Our knowledge about the morphological and genetic development of the lung is, however, highly biased towards amniotes, and consequently the original form of this evolutionary novelty among osteichthyans remains largely elusive. One hypothesis, formed and supported by studies on tetrapods (particularly mammals and birds), assumes that the lung evolved through a modification of the pharyngeal pouch (Kastschenko, 1887), as the lung bud develops at the pharyngo-oesophageal junction during embryonic development. Consequently, this view (Kastschenko, 1887; Kuratani and Tanaka, 1990) predicts that the primitive lungs appeared as bilaterally paired organs at the caudolateral part of the pharynx. Indeed, in embryology, lungs of living tetrapods have been mostly described as paired derivates of the respiratory tube, arisen from paired and small hollow swellings (Flint, 1906). Previous studies on amphibians have also proposed that the lung bud develop from paired rudiments of the ventral portions of the eighth pharyngeal pouches (Goodrich, 1931; Marcus, 1937; Perry et al., 2001). Additionally, the plesiomorphic state of lungs has been mostly described as paired organs (Funk et al., 2020). On the other hand, another hypothesis does not constrain the evolutionary origin of the lung to the serial homologue of the pharyngeal pouch (Greil, 1913; Neumayer, 1930; Wassnetzov, 1932). In this view, although the possibility that the primitive lung developed on the pharyngeal endoderm is not excluded, the primitive lung is considered to appear on the floor of the pharynx, or more generally, on the floor of the foregut. This scenario does not predict bilaterally paired forms of primitive lungs.

Curiously, some living vertebrates display an unpaired organ, leaving the ancestral condition equivocal. The sister group to all other extant actinopterygians, the obligate air-breathing polypterids (Icardo et al., 2017), breath air using lungs, which have previously been described as a paired organ (Icardo et al., 2017; Geoffrey Saint Hilaire, 1802; Graham, 1997). However, in adult specimens of Polypterus senegalus the glottis only opens to the right sac and the left sac is connected to the right sac by a separate opening (Graham, 1997), raising old questions about its true paired condition. Among sarcopterygians, the unpaired lung of coelacanths is unequivocal. The living coelacanth Latimeria chalumnae, that inhabits moderate deep-water and makes gas exchanges only through gills, have an unpaired lung with no outline of a second bud at different developmental stages (Cupello et al., 2015; Cupello et al., 2017a; Cupello et al., 2017b; Cupello et al., 2019). In the extant sister group of all tetrapods (Amemiya et al., 2013), namely lungfishes, the three extant genera have lungs capable to uptake oxygen from the air. However, in the facultative air-breather Neoceratodus forsteri, the lung is described as unpaired in adults (Greil, 1913; Graham, 1997; Grigg, 1965). In contrast with both South American and African lungfishes, the Lepidosirenoidea, that are obligated air-breathers and have a lung described as a ventral paired organ (Graham, 1997) like tetrapod lungs.

To follow lung evolutionary history in vertebrates, we analyzed the morphogenesis of lungs of key living osteichthyans (Figures 1 and 2, Figures 3 and 4, Figures 5 and 6). Embryos, larvae, juveniles and adults of P. senegalus, N. forsteri, Lepidosiren paradoxa were examined. To compare the lung anatomy of osteichthyan fishes with tetrapods, we studied also an extensive developmental series of the living salamandrid Salamandra salamandra, from early and late larvae to juveniles before and after metamorphosis (Figure 5). As salamanders are often considered to have retained plesiomorphic characteristics of tetrapod stance and locomotion (Pierce et al., 2020), we used them here also as a model for understanding lung evolution in tetrapods. Specimens of mentioned taxa were examined through x-ray microtomography, the unique effective non-invasive methodology to study their morphology and histology at a three-dimensional (sub) microscale. When possible, we proceeded also with dissections and the study of histological sections. We compare our results with the available information from the lung of fossil taxa, the coelacanths and salamanders (Cupello et al., 2019; Brito et al., 2010; Tissier et al., 2017).

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Traditionally, vertebrate lungs are defined as ventral paired organs derived from the ventral portion of the posterior pharynx or post-pharyngeal foregut (Perry et al., 2001; Funk et al., 2020; Lambertz et al., 2015; Graham, 1997; Kardong, 2015). However, we demonstrate here the presence of an unambiguous unpaired lung, that develops from the ventral foregut, but sometimes occupy the dorsal position later in the development of osteichthyan fishes (Figure 7). Based on extensive developmental series of different vertebrate taxa, we present a new interpretation of some lungs previously considered as paired, and therefore, a new definition of paired lungs. Based on our results, true paired lungs are stated when bilateral lung buds arise simultaneously and are both connected directly to the foregut, as observed in the salamander (Figures 5B, D, 7).

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The sister group to all other living actinopterygian (polypterids) and all living sarcopterygian fishes have a clearly unpaired lung in early developmental stages (Figure 1B) that can be developed in later stages either in a unilobed or a secondarily multilobed lung (Figures 1D–F ,–7), but not in a true paired lung. P. senegalus and the lungfish L. paradoxa possess secondary multilobed structure from the larval stage onward since the lung is derived from a unilateral connection to the foregut (Figure 4G, H, J and K). The presence of this secondary multilobed morphology likely represents an advantage for the obligatory air-breathing behavior of these taxa, raising the respiratory compliance. In the teleost Batrachomoeus trispinosus, the non-respiratory gas bladder is also described as paired (Rice and Bass, 2009), although it is most probably a secondary condition.

The most parsimonious scenario inferred from our data mapped on the phylogenetic framework (Figure 7) is that the vertebrate lung was unpaired at the evolutionary origin. Since soft tissues are rarely preserved in fossils, living lunged osteichthyans are key taxa for the understanding of how evolutionary constraints shaped breathing adaptations on land. Our study suggests that the ancestral condition of the lung is a median unpaired organ (Figure 7), thereby being inconsistent with the scenario that the lung evolved through a modification of the posteriormost pharyngeal pouch assumed to be present in primitive taxa (Kastschenko, 1887; Goodrich, 1931). Consequently, the evolutionary origin of the lung was likely independent of the pharyngeal pouch at the morphological level.

From this evolutionary point of view, our results lead to a new definition of the vertebrate lung: either an unpaired or paired respiratory organ developing ventrally from the foregut. Vestigial forms secondarily released from the respiratory function should be also designated as lungs (e.g. the lung of coelacanths). Some criteria previously used for discriminating lungs from gas bladders are no longer supported, including: paired/unpaired organization, position ventral to the alimentary tract (Marcus, 1937; Funk et al., 2020; Lambertz et al., 2015; Graham, 1997), as well as its function. The dorsal position of the majority of osteichthyans lungs described here may be related to its dual and secondary functionality of respiration and buoyancy control (Thomson, 1968). Actually, the only morphological characteristic that can be used to distinguish lungs and gas bladders is the ventral and dorsal origins from the foregut, respectively (Funk et al., 2020; Cass et al., 2013). This phenotypic differentiation into true paired lungs in tetrapods may be related to differential gene expressions (Funk et al., 2020; Bi et al., 2021). Nevertheless, at a level of developmental mechanism, the possibility of co-options of gene regulatory networks of the pharyngeal pouch morphogenesis cannot be excluded, as both the lung bud and pharyngeal pouch develop through the invagination of the foregut endoderm. Our results open the door for future molecular analyses to trace possible regulatory elements for the evolutionary transition from unpaired lungs to true paired lungs in tetrapods.

According to morphological evidence presented here, bifurcation morphogenesis into true paired lungs was not developed yet in osteichthyan fish ancestors. Based on the extant phylogenetic bracket, we infer that the bilaterally paired nature of the lung evolved only in the lineage towards fossil and extant tetrapods, as a synapomorphy of this clade (Figures 5 and 7). This morphological modification brought about improvement of the efficiency in oxygen-uptake from the air, as the paired lungs having parallel air flows exchange the air more quickly than the unpaired lung having only single air flow does. This innovation led to the elevation of metabolic rate that was required for the sustained body support against the gravity. Paired lungs may have been present also in early tetrapods and were probably essential to raise lung surface area and volume capacity during the evolution of vertebrate respiratory system and the air-breathing intensification at the water-to-land transition.

All data used in this work are publicly available via Dryad https://doi.org/10.5061/dryad.vdncjsxx1.

1. 1. Cupello C

   (2022) Dryad Digital Repository

   Data from: Lung evolution in vertebrates and the water-to-land transition.

   https://doi.org/10.5061/dryad.vdncjsxx1

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Author details

1. Camila Cupello

   Departamento de Zoologia-IBRAG, Universidade do Estado do Rio de Janeiro, Rio de Janeiro RJ, Brazil

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   camila.dc@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6030-8670

2. Tatsuya Hirasawa

   Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, Tokyo, Japan

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6868-3379

3. Norifumi Tatsumi

   Department of Anatomy, The Jikei University School of Medicine, Tokyo, Japan

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6640-150X

4. Yoshitaka Yabumoto

   Kitakyushu Museum of Natural History and Human History, 2-4-1 Higashida, Yahatahigashi-ku, Kitakyushu, Fukuoka, Japan

   Contribution

   Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4736-8246

5. Pierre Gueriau

   1. Institute of Earth Sciences, University of Lausanne, Lausanne, Switzerland
   2. Université Paris-Saclay, CNRS, ministère de la Culture, UVSQ, MNHN, Institut photonique d’analyse non-destructive européen des matériaux anciens, Saint-Aubin, France

   Contribution

   Data curation, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7529-3456

6. Sumio Isogai

   Department of Anatomy, Iwate Medical University School of Medicine, Iwate, Japan

   Contribution

   Data curation, Methodology, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Ryoko Matsumoto

   Kanagawa Prefectural Museum of Natural History, Kanagawa, Japan

   Contribution

   Data curation, Methodology, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0334-7259

8. Toshiro Saruwatari

   1. Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, Japan
   2. Seikei Education and Research Center for Sustainable Development, Tokyo, Japan

   Contribution

   Data curation, Funding acquisition, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5761-8528

9. Andrew King

   Synchrotron SOLEIL, L’orme des Merisiers Saint-Aubin, Gif-sur-Yvette Cedex, France

   Contribution

   Methodology, Software, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8542-1354

10. Masato Hoshino

    Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), Hyogo, Japan

    Contribution

    Methodology, Software, Validation, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4167-2706

11. Kentaro Uesugi

    Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), Hyogo, Japan

    Contribution

    Methodology, Software, Validation, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2579-513X

12. Masataka Okabe

    Department of Anatomy, The Jikei University School of Medicine, Tokyo, Japan

    Contribution

    Data curation, Investigation, Methodology, Validation, Writing – original draft, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8618-6859

13. Paulo M Brito

    Departamento de Zoologia-IBRAG, Universidade do Estado do Rio de Janeiro, Rio de Janeiro RJ, Brazil

    Contribution

    Conceptualization, Data curation, Funding acquisition, Methodology, Validation, Writing – original draft, Writing – review and editing

    For correspondence

    pbritopaleo@yahoo.com.br

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4853-8630

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