Lung Development of Monotremes: Evidence for the Mammalian Morphotype

Lung Development of Monotremes:Evidence for the Mammalian

MorphotypeKIRSTEN FERNER,1* ULRICH ZELLER,1 AND MARILYN B. RENFREE2

1Institute of Systematic Zoology, Museum of Natural History, Berlin, Germany2Department of Zoology, The University of Melbourne, Victoria, Australia

ABSTRACTThe reproductive strategies and the extent of development of neo-

nates differ markedly between the three extant mammalian groups: theMonotremata, Marsupialia, and Eutheria. Monotremes and marsupialsproduce highly altricial offspring whereas the neonates of eutherianmammals range from altricial to precocial. The ability of the newbornmammal to leave the environment in which it developed depends highlyon the degree of maturation of the cardio-respiratory system at the timeof birth. The lung structure is thus a reflection of the metabolic capacityof neonates. The lung development in monotremes (Ornithorhynchus ana-tinus, Tachyglossus aculeatus), in one marsupial (Monodelphis domes-tica), and one altricial eutherian (Suncus murinus) species was examined.The results and additional data from the literature were integrated into amorphotype reconstruction of the lung structure of the mammalian neo-nate. The lung parenchyma of monotremes and marsupials was at theearly terminal air sac stage at birth, with large terminal air sacs. Thelung developed slowly. In contrast, altricial eutherian neonates had moreadvanced lungs at the late terminal air sac stage and postnatally, lungmaturation proceeded rapidly. The mammalian lung is highly conservedin many respects between monotreme, marsupial, and eutherian speciesand the structural differences in the neonatal lungs can be explainedmainly by different developmental rates. The lung structure of newbornmarsupials and monotremes thus resembles the ancestral condition of themammalian lung at birth, whereas the eutherian newborns have a moremature lung structure. Anat Rec, 292:190–201, 2009. � 2008 Wiley-Liss,Inc.

Key words: lung development; Monotremata; Marsupialia;Eutheria; phylogenetic reconstruction

The three subclasses of living mammals are distin-guished by their modes of reproduction that haveevolved independently during the past 125 million years(Tyndale-Biscoe and Renfree, 1987). Monotremes layeggs and incubate them for the last third of their embry-onic development, and yet being true mammals, theyhave hair and suckle their young (Griffiths, 1968, 1978).The body dimensions and external morphology of newlyhatched monotremes resemble that of newbornmarsupials (Fig. 1A). In contrast to the Monotremata,Marsupialia and Eutheria represent two viviparousevolutionary lines of the Mammalia. The young of

*Correspondence to: Kirsten Ferner, Institute of SystematicZoology, Museum of Natural History, Berlin, Germany.E-mail: [email protected]

Grant sponsor: Deutsche Forschungsgemeinschaft (DFG);Grant number: GRK 503; Grant sponsor: Berliner Programmzur Forderung der Chancengleichheit von Frauen.

Received 27 March 2008; Accepted 21 August 2008

DOI 10.1002/ar.20825Published online 2 December 2008 in Wiley InterScience (www.interscience.wiley.com).

� 2008 WILEY-LISS, INC.

THE ANATOMICAL RECORD 292:190–201 (2009)

marsupials are born after a brief gestation period at avery early stage of development and much of the devel-opment occurs postnatally in the pouch, while they areattached to one of the maternal teats (Fig. 1B) (Tyndale-Biscoe and Renfree, 1987; Tyndale-Biscoe, 2001; Zelleret al., 2001; Renfree, 2006). Newborn Eutheria, on theother hand, feature a wide range of developmentalstages, from altricial to precocial development, but theyare always more advanced in development than marsu-pial neonates and undergo a rapid postnatal develop-ment (Fig. 1C) (Hughes and Hall, 1998; Zeller et al.,2001; Szdzuy, 2008; Szdzuy et al., 2008).However, mammals that lay eggs (monotremes), mam-

mals that give birth to living but incompletely developedyoung (marsupials), and mammals that give birth tocomparatively well-developed young (eutherians) mayhave descended from ancestors that once reproduced bymeans of a closed egg (Sharman, 1973). In this context,the extant monotremes with their ancestral mode ofreproduction are of special interest for the reconstruc-tion of a mammalian morphotype in terms of repro-duction and development. Whereas the modes of repro-duction and development of marsupials and eutheriansare relatively well understood, that of monotremes hasbeen less well studied (Griffiths, 1968, 1978; Renfree,1995; Temple-Smith and Grant, 2001). Because of thevery limited availability of early ontogenetic stages,there are only a few studies that deal with the embry-onic and postnatal development of monotremes (Semon,1894; Kuhn, 1971; Griffiths, 1978; Zeller, 1989; Hughesand Hall, 1998).In mammals, the different reproductive strategies and

the degree of development of neonates offer an interest-ing starting point for investigations. The neonate is oneof the most vulnerable stages in the mammalian life

cycle and is therefore of special interest. The hatchlingsof the Monotremata and the neonates of Marsupialiaand altricial Eutheria are small in size, naked, and com-pletely dependent on maternal care (Fig. 1). These typesof neonates are presumed to be characteristic of themammals in the Mesozoic (Lillegraven, 1979). The abil-ity of the newborn animal to survive depends on a num-ber of adaptations, including the degree of maturation ofthe respiratory system at the time of birth. In utero mar-supial and monotreme embryos have a low mass specificmetabolic rate so that diffusion from maternal tissueacross the placenta is adequate to meet the metabolicneeds, whereas eutherian embryos have a high meta-bolic rate supported by active placental gas transfer. Atbirth, however, the newborn’s respiratory apparatusmust be mature enough to take over the gas-exchangefunction, with the exception of very immature marsu-pials such as dasyurids, in which the skin offers anadditional site for gas exchange (Mortola et al., 1999;Mortola, 2001). Lung development in mammals is notcomplete at birth (Burri, 1974; Thurlbeck, 1975; Zeltnerand Burri, 1987). Postnatal lung development consists ofthree consecutive steps. In the first step, lung volumeincreases by simple expansion of the airspaces. This isfollowed by a phase of rapid increase in lung tissuemass accompanied by a dramatic expansion of the inter-nal surface area due to a process of subseptation of theexisting airspaces (alveolization). The third step consistsof a remodeling of the interalveolar walls via an increasein height and thinning, and the transformation of theseptal capillary system from a double- to a largely sin-gle-network structure (microvascular maturation)(Burri, 1974; Zeltner and Burri, 1987). The degree oflung maturity at birth varies considerably between spe-cies, reflecting their stage of development (Runciman

Fig. 1. Drawings of a newly hatched O. anatinus (Monotremata; redrawn from Hughes and Hall, 1998),a newborn M. domestica (Marsupialia), and a newborn S. murinus (Eutheria). The altrical monotreme andmarsupial neonates are born at an very early developmental stage (A, B), whereas the altricial eutherianneonate (C) is more advanced in development but still immature. Scale bar, 5 mm.

191LUNG DEVELOPMENT IN MONOTREMES

et al., 1996). The lung of marsupials at birth is structur-ally immature, yet is mostly functional as a gasexchanger (Baudinette et al., 1988; Makanya et al.,2007). At birth, the lung in some marsupial species is atthe canalicular stage of development and though lungdiffusion and metabolic rate are strongly correlated, theallometric exponent suggests that smaller newbornshave relatively smaller diffusing capacity with respect totheir demand for oxygen (Frappell and MacFarlane,2006). Without improvement in functional or structuralparameters, newborn marsupials are reliant to varyingdegrees on skin gas exchange to compensate for theimmaturity of the lung (Mortola et al., 1999; Frappelland Mortola, 2000; Frappell and MacFarlane, 2006). Incontrast, in eutherians, the lung is more developed atbirth (Burri, 1974). The structural development of thelung in newborn eutherian mammals ranges from theterminal air sac stage in altricial species to the alveolarstage in precocial species (Szdzuy, 2008). In contrast tomarsupials and eutherians, the lung development ofmonotremes has not been investigated previously andthere are only a few studies describing the lung struc-ture of adult monotremes (Engel, 1962; Griffith, 1978;Perry et al., 2000). To reconstruct the mammalian lungmorphotype, the present histological study investigatedthe lung structure of pouch and burrow young monot-remes (echidna and platypus) and compared it to thelung development of a marsupial (gray short-tailed opos-sum) and a eutherian (musk shrew) species.

MATERIALS AND METHODS

Two platypus (Ornithorhynchus anatinus) and threeechidna (Tachyglossus aculeatus) young were availablefrom several collections at the Berlin Museum of Natu-ral History. In addition, the lungs of a plesiomorphicmarsupial species, the gray short-tailed opossum (Mono-delphis domestica, Didelphoidea), and of a plesiomorphiceutherian species, the musk shrew (Suncus murinus,Eulipothyphla), were investigated for the reconstructionof a mammalian respiratory morphotype. These taxawere selected because opossums and shrews are mostsuitable to represent basal marsupials and eutherians(Thompson and Nicoll, 1986; Kemp, 2005). The marsu-

pial and eutherian animals were taken from captive col-onies established at the animal facility of the Instituteof Systematic Zoology at the Berlin Museum of NaturalHistory. All sampling was approved by the AnimalExperimentation Ethics Committee of the Governmentof Berlin, Germany (registration number: G 0249/02).Details of the developmental stages and origin of theanimals examined for the lung development are pro-vided in Table 1.

Light Microscopy

The monotremes available from the Hill and Wilsoncollection were already prepared for light microscopy(serially sectioned in a transverse plane, mounted onglass slides and stained). In addition, lungs of Monodel-phis domestica and Suncus murinus were processed forhistology. The whole torso (for neonates or early post-natal stages) and the isolated lungs (at later ontogeneticstages) were fixed by perfusion through the superiorvena cava via the infundibulum of the right ventricle ofthe pulmonary circulation. The blood vessels were rinsedwith Ringer’s solution and fixed with Bouin’s solution.In addition, to ensure good fixation for the entire lungtissue, the fixing agent was instilled in the trachea.Finally, the torsos or the isolated lungs were immersedin fixative and after 1 day fixing in Bouin’s the speci-mens were rinsed in 70% ethanol. Torsos that containedbones were decalcified for 4 days with a stock solution(100 mL dimethylsulfoxide, 15 mL nitric acid (65%)),rinsed in sodium sulphate solution (5%) and distilledwater and transferred to 70% ethanol before automaticparaffin embedding (Shanon Hypercenter XP, Life Sci-ence International GmbH, Frankfurt, Germany). Thelungs were serially sectioned in a transverse plane at 7–10 mm and stained with Azan, trichrome, or hematoxylinand eosin (HE). All series were viewed in a stereomicro-scope (Leica MZ 12; Wildt, Switzerland) in transmittedlight.

Morphometric Measurements

A modified method of Weibel (1963) was used to mea-sure the mean airspace diameter and mean septumthickness. First, for each developmental stage, five

TABLE 1. Lung morphometry in two monotremes, one marsupial and one eutherian species

Species Stage CRL (mm) Specimen no.Mean air spacediameter (mm)

Mean septumthickness (mm)

Ornithorhynchus anatinus Neonatea 16.75 M44 320.1 6 13.1 29.4 6 0.7Burrow youngb 29.3 AMNH 201969 220.9 6 8.7 28.7 6 0.6

Tachyglossus aculeatus Embryoa 12.5 M158 – –Pouch younga 33 MO75 258.1 6 12.4 37.9 6 0.7Youngc 98 – 193.4 6 7.2 22.3 6 0.7

Monodelphis domestica Neonateb 10 86 453.3 6 21.9 45.4 6 1.88 ppb 13 89 162.7 6 6.3 40.9 6 1.128 pp 20 150 65.1 6 2.9 13.0 6 0.4

Suncus murinus Neonate 35 11 103.2 6 2.8 22.8 6 1.27 pp 62 36 64.8 6 1.8 11.7 6 0.714 pp 85 47 25.4 6 0.9 7.1 6 0.3

pp, days postpartum.aHill collection, Berlin Museum of Natural History.bProf. Zeller, Museum of Natural History Berlin.cWilson collection, Museum of Natural History Berlin.

192 FERNER ET AL.

randomly chosen digital photographs of the most periph-eral lung regions with the corresponding scale bar wereprinted on fixed co-ordinates. A graphics tablet con-nected to a minicomputer was calibrated with the scalebar and a line was randomly cast over different sectionsof the lung. On each digital photograph 20 airspace di-ameter or airspace septa intersecting with the line weremeasured with the graphics tablet, yielding a total of100 for each case. All group data are presented asmeans 6 1 SE.

Phylogenetic Reconstruction

To understand evolution, it is necessary to know notonly the character states of living organisms, but also oftheir ancestors. An increasingly popular method is toinfer ancestral character states by mapping the charac-ter states of living organisms onto phylogenies using themethod of maximum parsimony (Cunningham et al.,1998; Zeller and Freyer, 2001). As pointed out by Cun-ningham et al. (1998), the algorithm uses a ‘‘downpass’’and ‘‘uppass’’ traversal to optimize ancestral states usingtwo rules: Rule 1—if descendant nodes share any statesin common, assign the set of shared states to the ances-tor; Rule 2—if no states are shared in descendant nodes,assign the union of descendant’s states to ancestor. Thisprocedure was conducted using the systematics by West-heide and Rieger (2004).

RESULTS

The neonates of Ornithorhynchus anatinus, Monodel-phis domestica, and Suncus murinus represent therange of birth stages found in monotreme, marsupial,and altricial eutherian mammals (Fig. 1). The differen-ces between the tiny altricial monotreme and marsupialneonates (Fig. 1A,B) and the more developed but stillimmature altricial eutherian neonates (Fig. 1C) areclear. The newly hatched platypus was redrawn fromHughes and Hall (1998), who described a specimen fromthe Hill collection with a crown-rump length (CRL) of

16.75 mm, which is probably the same individual inves-tigated here for lung development. The newly hatchedplatypus was naked, had fused eyelids and mobile fore-limbs with five separated digits with blunt recurvedclaws but the immobile hindlimbs were paddles at amuch earlier stage. Features of the newborn platypuswere a prominent ‘‘yolk’’ navel, the presence of a bluntlyconical os caruncle and an egg tooth (Hughes and Hall,1998). It is of interest to note that the newly hatchedplatypus has an external form, apart from the latterthree features, reminiscent of a newborn marsupial (Fig.1A vs. B).The altricial eutherian neonate, although naked and

with closed eyes as well, was larger and had well-devel-oped extremities at birth (Fig. 1C).

Lung Structure of Ornithorhynchus anatinus

and Tachyglossus aculeatus (Monotremata)

Gross anatomy. The lungs of both monotreme spe-cies were asymmetrical, with the right lungs larger thanthe left lungs. In the lungs of Ornithorhynchus anatinusno fissures were apparent. However, the branching pat-tern of the lobar bronchi indicated a subdivision intopulmonary sections in both lungs. The right lung con-sisted of fused superior, middle, accessory (cardiac), andinferior lobes and the left lung had fused superior, mid-dle, and inferior lobes (Fig. 2A).The right lung of Tachyglossus aculeatus consisted of

completely separated superior, middle, accessory (car-diac), and inferior lobes. In the left lung there were nofissures, but the lobar bronchi indicated the presence offused middle and inferior lobes (Fig. 2B).

Histology. The lungs of the burrow- and pouchyoung Ornithorhynchus anatinus and Tachyglossus acu-leatus were at the early terminal sac stage of lung devel-opment (Figs. 3 and 4). The bronchial trees of the newlyhatched O. anatinus and the near-term embryo of T.aculeatus were poorly developed with cranially broad,

Fig. 2. Schematic representation of the bronchial tree of the lungs of Ornithorhynchus anatinus (A),Tachyglossus aculeatus (B), Monodelphis domestica (C), and Suncus murinus (D). 1, trachea. Right lung:2, superior lobe bronchus; 3, middle lobe bronchus; 4, accessory lobe bronchus; 5, inferior lobe bronchi.Left lung: 6, middle lobe bronchus; 7, inferior lobe bronchi; 8, superior lobe bronchus.

193LUNG DEVELOPMENT IN MONOTREMES

Fig. 3. Light micrographs of the lungs of a newly hatched (CRL:16.75 mm, A–D) and a burrow young (CRL: 29.3 mm, E–H) O. anati-nus. The lungs were at the early terminal sac stage of lung develop-ment. The broad main bronchi opened via smooth-walled channels inlarge terminal air sacs. The air sacs were separated by septa with adouble capillary network (arrows indicate capillaries). In the developing

platypus lung septal crests subdivided the large terminal air sacs. ap,pulmonary artery; as, air sac; b, bronchiole; be, bronchiolar epithelium;ds, double capillary septum; lb, lobar bronchus; mb, main bronchus;re, respiratory epithelium; sc, septal crest; swc, smooth-walled chan-nel. Azan staining. The scale bar indicates magnification.

194 FERNER ET AL.

Fig. 4. Light micrographs of the lungs of an embryonic (CRL: 12.5mm, A, B) and two pouch young (CRL: 33 mm, C–F; CRL: 98 mm, G,H) T. aculeatus. The lung of the near-term embryo revealed the pres-ence of large (however uninflated) terminal air sac and a double capil-lary septum (A, B). During the postnatal period, the bronchial treebecame ramified (C, G), but in the distal portions of the airwayssmooth-walled channels were still present (D). Numerous septal out-

growths subdivided the air sacs and gave them an irregular shape (E,F). The air sacs were separated by septa with a double capillary net-work (H, arrows indicate capillaries). as, air sac; b, bronchiole; bv,blood vessel; lb, lobar bronchus; mb, main bronchus; ds, double cap-illary septum; sc, septal crest; swc, smooth-walled channel. Azanstaining. The scale bar indicates magnification.

195LUNG DEVELOPMENT IN MONOTREMES

tapering main bronchi (Figs. 3A and 4A). The mainbronchi were lined with a one or two layered cuboidalciliated epithelium. Beneath a layer of three or foursmooth muscle cells stabilized the bronchus. Extrapul-monary cartilage supported the bronchial walls but thisdisappeared when the main bronchi entered the lung.Several short lobar bronchi branched off from the mainbronchi. They consisted of cuboidal epithelium and alayer of one or two smooth muscle cells and opened instructurally similar bronchioles (Fig. 3D). These gaverise to smooth-walled channels, lined with respiratoryepithelium (type I and type II pneumocytes) that openeddirectly in the large terminal air sacs (Fig. 3A). Large airsacs were present for gaseous exchange, but had not yetinflated in the embryonic T. aculeatus (Figs. 3B and 4B).In the lung of the newly hatched O. anatinus, the air-

spaces measured 320.1 6 13.1 mm on average. Theywere lined with respiratory epithelium and appearedsmooth-walled with only a few septal crests protrudingfrom the septa (Fig. 3B,C). The extensive septa separat-ing the air sacs consisted of a double capillary network(Figs. 3C and 4B). In the newly hatched O. anatinusthese septa had a thickness of 29.4 6 0.7 mm.During the postnatal development, the large air sacs

became more and more subdivided by septal crests anddecreased in size (Figs. 3E–G and 4E,F). The burrowyoung O. anatinus (CRL: 29.3 mm) examined here stillhad an immature lung structure with an average air-space size of 220.9 6 8.7 mm and a septum thickness of28.7 6 0.6 mm. The septa consisted of a double capillarynetwork (Fig. 3H). There was a developed bronchial treein the earliest postnatal stage of T. aculeatus (CRL:33 mm). It consisted of main, lobar, and segmental bron-chi and bronchioles. The terminal airways consisted ofbronchioles, which gave rise to smooth-walled channelsthat opened into the air sacs (Fig. 4C,D). Numerous sep-tal crests protruded from the septa into the air saclumen and gave them an irregular shape (Fig. 4E).Although the septal outgrowths caused subdivisions, theair sacs were still large with an average airspace size of258.1 6 12.4 mm. All newly formed septal crests as wellas the older septa were composed of a double capillarynetwork (Fig. 4F). They had a mean thickness of 37.9 60.7mm. In the latest postnatal stage of T. aculeatus(CRL: 98 mm) the bronchial tree appeared well devel-oped with bronchioles extending far to the periphery ofthe lung (Fig. 4G). Because of a further subdivision ofthe lung parenchyma, the air spaces decreased in size to193.4 6 7.2 mm. The septa separating the air sacsbecame thinner as well and measured 22.3 6 0.7 mm onaverage. However, all septa in the lung of T. aculeatuscontained a double capillary network at this stage of de-velopment (Fig. 4H).

Lung Development of Monodelphis

domestica (Marsupialia)

Gross Anatomy. The lungs of Monodelphis domes-tica were asymmetrical. The right lung consisted of fourseparated pulmonary lobes (superior, middle, accessory(cardiac), and inferior lobes). The left lung appeared tobe unlobulated, but the branching pattern of lobar bron-chi indicated a fusion of middle and inferior lobes (Fig.2C).

Histology. In the newborn Monodelphis domestica,the lungs were at the early terminal air sac stage oflung development and consisted of a primitive system ofbranching airways that terminated in a number of largeterminal air sacs. The large terminal air sacs had alumen of 453.3 6 21.9 mm in diameter in Monodelphisdomestica (Fig. 5A). The air sacs were lined with a sim-ple squamous sheet of epithelium on top of an extensivecapillary bed. Cuboidal type II pneumocytes were inter-spersed between the simple squamous type I pneumo-cytes. Each air sac had its own capillary bed that wasseparated from the capillary bed of the adjacent air sac.The thickness of the septa located between the air sacswere on average about 45.4 6 1.8 mm in Monodelphisdomestica. During the early postnatal phase, the largeair sacs became more and more subdivided by septalcrests and decreased in size. At 8 days postpartum (pp),the air sacs measured 162.7 6 6.3 mm in diameter andthe double capillary septa were 40.9 6 1.1 mm thick(Fig. 5C). At this time, new air sacs developed at themost distal parts of the bronchial system. The firstalveoli, identified by the presence of single capillarysepta, were found at 28 pp in Monodelphis domestica(Fig. 5E). In addition, transformations in the distal partsof the terminal bronchioles took place. First respiratorybronchioles developed. They were characterized by flat-tened epithelium with shallow depressions in theirwalls. At this time, the average airspace diametersmeasured 65.1 6 2.9 mm in Monodelphis domestica.With alveolization, the transformation from double capil-lary septa into single capillary septa started. Comparedwith the earlier developmental stages, the thickness ofthe airspace septa was decreased remarkably to 13.0 60.4 in width.

Lung Development of Suncusmurinus (Eutheria)

Gross Anatomy. The lungs of Suncus murinuswere highly asymmetrical. The right lung of Suncusmurinus was divided into completely separate superior,middle, accessory (cardiac), and inferior lobes. A peculi-arity of the Suncus murinus lung was the branching ofthe right superior lobe bronchus directly from the tra-chea. In the left lung no fissures were present and thebranching pattern of lobar bronchi indicated the pres-ence of a small single inferior lobe only.

Histology. The lungs of the altricial neonate ofSuncus murinus were at the late terminal sac stage oflung development (Fig. 5B). At birth the conducting air-ways occupied a large portion of the lung volume andthe ramified bronchial tree extended far to the peripheryof the lung. The terminal air sacs in the lung of the neo-nate were numerous and small in size. They measured103.2 6 2.8 mm in diameter. The air sacs were linedwith squamous type I pneumocytes with occasionallyinterspersed type II pneumocytes, similar to those of themarsupial lung. The average thickness of the doublecapillary septa separating the air sacs was 22.8 6 1.2mm in the newborn Suncus murinus. The double capil-lary septa contained a central interstice of connectivetissue flanked on both sides by capillaries.The postnatal lung development proceeded very fast

in Suncus murinus. The first signs of the transformation

196 FERNER ET AL.

Fig. 5. Light micrographs of the lungs of Monodelphis domestica(neonate (A), 8 pp (C), 28 pp (E)) and of Suncus murinus (neonate (B),7 pp (D), 14 pp (F)). The lungs of the newborn marsupial M. domesticawere at the early terminal sac stage of lung development with large airsacs, separated by thick double capillary septa (A). During the slowpostnatal lung development, the air sacs became smaller in size (C),but alveoli were not present until the age of 4 weeks (E). The lungs ofthe newborn altricial eutherian Suncus murinus were at the late termi-

nal sac stage of lung development (B). The postnatal lung develop-ment was fast and alveoli and associated structures were present at7 pp (D). A highly subdivided lung parenchyma was found at 14 pp(F). as, air sac; b, bronchiole; br, respiratory bronchiole; bt, terminalbronchiole; lb, lobar bronchus; mb, main bronchus; sa, alveolar sac;sc, septal crest; swc, smooth-walled channel. Azan staining (A, C, E);Hematoxylin and Eosin staining (B, D); Trichrome staining (F). Thescale bar indicates magnification.

197LUNG DEVELOPMENT IN MONOTREMES

TABLE 2. Characters of the mammalian morphotype (0) and apomorphic characters (I)

Character no.

Characterstate 0

(morphotype) Taxon

Characterstate I

(apomorphy) Taxon

1 Low birth weight (<1 g) Tachyglossus aculeatus High birthweight (>1 g)

Suncus murinusOrnithorhynchus.anatinus Sorex vagrans (2)Monodelphis domestica Mus musculus (2)Didelphis virginiana (1) Mesocricetus auratus (2)Isoodon macrourus (1) Rattus rattus (2)Trichosurus vulpecula (1)Macropus eugenii (1)

2 Closed eyes at birth Tachyglossus aculeatusOrnithorhynchus.anatinusMonodelphis domesticaDidelphis virginiana (1)Isoodon macrourus (1)Trichosurus vulpecula (1)Macropus eugenii (1)Suncus murinusSorex vagrans (2)Mus musculus (2)Mesocricetus auratus (2)Rattus rattus (2)

3 Lack of pelage at birth Tachyglossus aculeatusOrnithorhynchus.anatinusMonodelphis domesticaDidelphis virginiana (1)Isoodon macrourus (1)Macropus eugenii (1)Suncus murinusSorex vagrans (2)Mus musculus (2)Mesocricetus auratus (2)Rattus rattus (2)Trichosurus vulpecula (1)

4 Lung at air sac stageat birth

Tachyglossus aculeatusOrnithorhynchus.anatinusMonodelphis domesticaDidelphis virginiana (3)Isoodon macrourus (4)Trichosurus vulpecula (5)Macropus eugenii (6)Suncus murinusSorex vagrans (7)Mus musculus (8)Mesocricetus auratus (9)Rattus rattus (10)

5 Large terminal airsacs at birth

Tachyglossus aculeatus Small terminal airsacs at birth

Suncus murinusOrnithorhynchus.anatinus Sorex vagrans (7)Monodelphis domestica Mus musculus (8)Didelphis virginiana (3) Mesocricetus auratus (9)Isoodon macrourus (4) Rattus rattus (10)Trichosurus vulpecula (5)Macropus eugenii (6)

6 Thick double capillaryseptum at birth

Tachyglossus aculeatus Thin double capillaryseptum at birth

Suncus murinusOrnithorhynchus.anatinu Mesocricetus auratus (9)Monodelphis domestica Rattus rattus (10)Didelphis virginiana (3)Isoodon macrourus (4)Trichosurus vulpecula (5)Macropus eugenii (6)

7 Late formation of alveoli Monodelphis domestica Early formationof alveoli

Suncus murinusDidelphis virginiana (3) Mus musculus (8)Isoodon macrourus (4) Mesocricetus auratus (9)Trichosurus vulpecula (5) Rattus rattus (10)Macropus eugenii (6)

Species given in bold were investigated in this study. References for data: (1) Tyndale-Biscoe and Renfree (1987), (2) Pusch-mann (2004), (3) Krause and Leeson (1975), (4) Gemmell (1986), (5) Buaboocha and Gemmell (1997), (6) Runciman et al.(1996), (7) Foresman (1994), (8) Ten Have-Opbroek (1980), (9) Szdzuy (2008), (10) Burri (1974).

198 FERNER ET AL.

from the terminal sac stage to the alveolar stage wereobserved 4 days after birth in Suncus murinus. Thenewly formed single capillary septa consisted of only onecentrally located capillary bed abutting on both sides onthe adjacent airspace. They separated the newly formedalveoli. In contrast to earlier developmental stages, re-spiratory bronchioles were present at 7 pp (Fig. 5D).These structures are characteristic of the alveolizedlung. At 14 days the septation had progressed further.The bronchial tree was widely ramified and respiratorybronchioles were common in the lung (Fig. 5F).

DISCUSSION

The lungs of mammals, including that of the monot-remes, are different from those of Aves and Reptilia inmany ways, but principally in the development of abranching treelike system of intrapulmonary bronchi(Griffiths, 1978). There are no previous studies on lungdevelopment in monotremes and only little informationabout the lung structure of adult platypus (Engel, 1962)and echidna (Engel, 1962; Griffiths, 1978; Perry et al.,2000). Engel (1962) described the lungs of both speciesas ‘‘primitive’’ mammalian lungs, with the typical acinarstructure, but Perry et al. (2000) concluded that the re-spiratory system of the adult echidna differed in no fun-damental way from that seen in other mammals.Our comparison of the monotreme, marsupial, and

eutherian lung structure showed that the mammalianlung is conserved in many respects. The asymmetry ofright and left lungs, including the presence of an acces-sory (cardiac) lobe in the right lung, is common in manydistantly related mammalian groups and seems to beplesiomorphic for Mammalia (Nakakuki, 1980). Thepresence of a superior lobe bronchus in the left lungs ofOrnithorhynchus anatinus and of some eutherian mam-mals (e.g. Tupaia belangeri, Macroscelides proboscideus,Equus caballus) suggests the original presence of supe-rior, middle, and inferior lobes in the left lung of themammalian morphotype (Nakakuki, 1980; Szdzuy,2008). However, in many mammalian species (e.g.Tachyglossus aculeatus, Monodelphis domestica, Macro-pus eugenii, and Mesocricetus auratus) the superior lobebronchus is missing and only middle and inferior lobes(often fused) are present (Szdzuy, 2008).The morphogenetic processes of alveolization and sep-

tal maturation in monotremes, marsupials, and euther-ians are similar from one species to the other, but thetiming is different. The earliest stage of lung develop-ment at birth seems to be the transition from canalicu-lar to early terminal sac stage, because a gas exchangearea consisting of a capillary septum with a blood-airbarrier must be present. This developmental stage withlarge terminal air sacs can be found in newborn Marsu-pialia and newly hatched Monotremata. The lung diffu-sion in newborn marsupials is less than that observed inneonatal eutherians of similar mass. It has been attrib-uted to poor surface area due to septal connective tissueintervening between the alveolar epithelium and theendothelium of capillaries over part of the air sac(Runciman et al., 1998). During the slow postnatal de-velopment, the large air sacs become subdivided andseparated by septal crests. The air sacs decrease in size.It takes much longer for marsupials to reach the samedevelopmental stage found in newborn eutherians, for

example Monodelphis domestica reaches the small airsac stage, already present in Suncus murinus at birth,with 21 days. Because of the rarity of available materialit is impossible to track the lung development of monot-remes in detail, but our data suggest that lung develop-ment in monotremes is as slow as in marsupials. AsZeltner and Burri (1987) pointed out, the structural hall-marks of postnatal lung development are twofold. Theyconsist first in a subseptation of the existing airspacesinto smaller units, the alveoli, and second, in the remod-eling of the septal, and particularly the microvascularstructure. The former process results in a rapid increasein the internal surface area of the lung, and the lattertransforms the double-layered into a single-layered cap-illary network (Zeltner and Burri, 1987). Lung develop-ment is characteristically slow in marsupials (Krauseand Leeson, 1975; Gemmell, 1986; Buaboocha and Gem-mell, 1997; Burri et al., 2003) and the formation ofalveoli and microvascular remodeling does not startbefore 28 pp in Monodelphis domestica. In altricialeutherians, postnatal lung development proceeds veryfast and the first alveoli appear within a few days ofbirth (Burri, 1974; Ten Have-Opbroek, 1980). In Suncusmurinus 4 pp, the first alveoli and single capillary septaare present. Although the onset of alveolization isunknown in monotremes, a typical bronchioalveolarlung is present in the adult echidna (Perry et al., 2000).However, a peculiarity was found in the adult echidna,suggesting that the microvascular remodeling does notoccur completely in the echidna lung. Beside single cap-illary septa, a double capillary net is present over largeportions of the alveolar surface (Perry et al., 2000). Thisfeature is unusual for the adult mammalian lung, wherenormally only single capillary septa are present. Physio-logically, the double capillary net provides a high capil-lary loading, that is, a large pulmonary blood volumeper unit gas-exchange area, which is considered as ad-vantageous for an animal with a low overall breathingfrequency like the echidna (Perry et al., 2000). But theechidna is not the only mammal with a double capillarynet in the adult lung. It seems to be convergent with thelung structure in some diving eutherian mammals, andat least one group of fossorial rodents (Bathyergidae),which also demonstrate these structures (Wislocki andBelanger, 1940; Wallau, 1998). The double capillary netis believed to be ancestral in monotremes but derived indiving mammals (Engel, 1962). But taking into consider-ation that it can be found in very different groups ofmammals, it could also be derived in general as an adap-tation to similar physiological demands (e.g., breath-holding at diving or burrowing way of life) (Perry et al.,2000).In general, the pulmonary structure at birth depends

on the extent of intrauterine development (Engel, 1962).Thus, the high degree of immaturity in the lungs ofnewborn marsupials and monotremes reflect their repro-ductive modes with a general immaturity at birth. Incontrast, eutherians have a more advanced lung atbirth, reflecting the longer intrauterine developmentthat leads to more developed neonates. It seems to belikely that marsupials and monotremes represent theoriginal degree of development of the mammalian lungat birth, whereas the eutherian newborns are derived toa lesser (altricial) of greater (precocial) degree. Thus theancestral characters present in the mammalian morpho-

199LUNG DEVELOPMENT IN MONOTREMES

type and those that are derived are summarized in Ta-ble 2. They are based on the character distributionwithin the four mammalian species examined and addi-tional literature for marsupial and altricial eutherianspecies (Fig. 6). The neonate in the mammalian morpho-type, represented by Ornithorhynchus anatinus, Tachy-glossus aculeatus, and Monodelphis domestica, wouldhave been born naked with a low birth weight of lessthan 1 g, and with closed eyes. The neonatal lung wouldbe at the air sac stage of lung development with largeterminal air sacs, which are separated by thick doublecapillary septa. Probably, a late formation of alveoli ischaracteristic for the mammalian morphotype. In con-trast to monotremes and marsupials, the eutherian neo-nate is derived in some characters. The neonate of altri-cial eutherians has a greater birth weight relative tomarsupials of more than 1 g, but is naked and also hasclosed eyes. The lung is at the air sac stage of lung de-velopment with small terminal air sacs, separated bythin double capillary septa. In altricial eutherians, theformation of alveoli begins shortly after birth. The mor-photype reconstruction of the mammalian neonatal lungsuggests that the respiratory apparatus was once a rela-

tively simple structure. Thus the lung structure ofnewborn marsupials and monotremes resembles the an-cestral condition of the mammalian lung at birth,whereas the eutherian neonates have a more maturelung structure.

ACKNOWLEDGMENTS

We thank Mrs. Jutta Zeller for the technical supportwith the histological processing of the material. MBRwas supported by an Australian Research Council Fed-eration Fellowship.

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