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. 2014 Nov 14;226(1):1–12. doi: 10.1111/joa.12255

Extreme variation in the atrial septation of caecilians (Amphibia: Gymnophiona)

Desiderius M de Bakker 1, Mark Wilkinson 2, Bjarke Jensen 1,3
PMCID: PMC4313894  PMID: 25400089

Abstract

Caecilians (order Gymnophiona) are elongate, limbless, snake-like amphibians that are the sister-group (closest relatives) of all other recent amphibians (frogs and salamanders). Little is known of their cardiovascular anatomy and physiology, but one nearly century old study suggests that Hypogeophis (family Indotyphlidae), commonly relied upon as a representative caecilian species, has atrial septation in the frontal plane and more than one septum. In contrast, in other vertebrates there generally is one atrial septum in the sagittal plane. We studied the adult heart of Idiocranium (also Indotyphlidae) using immunohistochemistry and confirm that the interatrial septum is close to the frontal plane. Additionally, a parallel right atrial septum divides three-fourths of the right atrial cavity of this species. Idiocranium embryos in the Hill collection reveal that atrial septation initiates in the sagittal plane as in other tetrapods. Late developmental stages, however, see a left-ward shift of visceral organs and a concordant rotation of the atria that reorients the atrial septa towards the frontal plane. The gross anatomies of species from six other caecilian families reveal that (i) the right atrial septum developed early in caecilian evolution (only absent in Rhinatrematidae) and that (ii) rotation of the atria evolved later and its degree varies between families. In most vertebrates a prominent atrial trabeculation associates with the sinuatrial valve, the so-called septum spurium, and the right atrial septum seems homologous to this trabeculation but much more developed. The right atrial septum does not appear to be a consequence of body elongation because it is absent in some caecilians and in snakes. The interatrial septum of caecilians shares multiple characters with the atrial septum of lungfishes, salamanders and the embryonic septum primum of amniotes. In conclusion, atrial septation in caecilians is based on evolutionarily conserved structures but possibly exhibits greater variation than in any other vertebrate order.

Keywords: cardiovascular morphology, development, evolution, heart, septum, vertebrate

Introduction

Dipnoi, represented by extant lungfishes, are the only fishes with partial structural division of the heart into pulmonary venous (left) and systemic venous (right) sides (Jensen et al. 2010; Jensen & Moorman, 2015) but it is not known whether this structural division is homologous with septation of the tetrapod heart. Studies on the structure and development of cardiac septation in extant amphibians (frogs, salamanders and caecilians) are therefore important for identifying the earliest characters of cardiac septation in tetrapods. In the African and South American lungfishes, the division is by the connective tissue of the pulmonary fold and atrioventricular plug that connects to the muscular septa in the atrium and ventricle and then by connective tissue ridges in the outflow tract (Robertson, 1913; Icardo et al. 2005a,b). The Australian lungfish has little more than the pulmonary fold, atrioventricular plug and modified outflow tract valves, all of connective tissue, to separate left and right sides of the heart (Klitgaard, 1978). These structures derive from embryonic mesenchyme and in early development the configuration of mesenchyme in the heart of lungfishes and birds and mammals is strikingly similar (Benninghoff, 1933; Jensen & Moorman, 2015).

The hearts of amphibians typically comprise five myocardial cavities; the sinus venosus, the left and right atrium, the ventricle and the conus arteriosus (Jensen et al. 2014b). Only the atrial and ventricular cavities have trabeculations and in the frog Xenopus these trabeculations have similar gene expression, for instance Nppa (Jensen et al. 2012). Nppa is expressed in a similar way in the mammalian embryonic heart but atrial trabeculations of the mammalian heart are normally referred to as pectinate muscles. In amphibians generally, there is a prominent atrial septum but little, if any, ventricular septation (Putnam & Dunn, 1978; Jensen et al. 2014b). Atrial septation in amphibians is much more varied than in amniotes (reptiles, birds and mammals), which have a full interatrial septum oriented in the sagittal plane. In frogs and toads (order Anura, Fig. 1) the interatrial septum is always full and oriented as in amniotes (Benninghoff, 1933; Ramsdell et al. 2006). The interatrial septum of salamanders (order Caudata, Fig. 1) may be full, partial and perforated or all but gone in certain lungless species and may be positioned approximately 45° towards the frontal plane (Benninghoff, 1933; Putnam & Kelly, 1978). Caecilians (order Gymnophiona, a small group of about 200 species mainly restricted to the tropics and notoriously understudied) are elongate, limbless, snake-like amphibians that are either fossorial or aquatic (see Wilkinson, 2012) and currently comprise 10 families (Wilkinson et al. 2011; Kamei et al. 2012; see Fig. 1). The literature on their cardiovascular anatomy is mostly old accounts with infrequent illustrations. It is clear that the heart is very elongate and has the same cavities as in other amphibians. The interatrial septum may be in the sagittal plane or, as seen in the family Typhlonectidae, in the frontal plane (Wilkinson & Nussbaum, 1997). One study from 1935 reports the presence of multiple atrial septa in Hypogeophis rostratus (family Indotyphlidae) oriented in the frontal plane (Schilling, 1935).

Fig. 1.

Fig. 1

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Caecilian families in the tetrapod phylogeny. Amphibians comprise three extant orders with Anura (frogs and toads) and Caudata (salamanders) most closely related (comprising the group Batrachia) and Gymnophiona (caecilians) originating from the most basal divergence within the group. Amniotes (reptiles, birds and mammals) share an ‘amphibian’ ancestry. In black are the names of the investigated groups of this study.

Here, we document atrial septation in adult Idiocranium, a caecilian genus that is closely related to Hypogeophis (both are in the Indotyphlidae), where atrial septation appears highly specialised. We also report aspects of cardiac development of Idiocranium, based on specimens of the Hill collection. Additionally, we interpret in a phylogenetic context literature reports and new observations of the gross morphology of atria of representatives of seven of the 10 extant caecilian families. Our findings are further compared with other classes of vertebrates, including snakes, to study the effect of, for instance, body elongation on atrial septation.

Materials and methods

Animals

Due to its close relationship to Hypogeophis (and the unavailability of the latter), we investigated hearts in an undescribed species (see Gower et al. 2014) of Idiocranium (n = 3, body mass 3.19 g ± 0.43 (SD); specimen specific code (ssc) MW9511–MW9513). Animals were anaesthetised in MS-222 (2 g L−1) and their hearts excised, fixed overnight in 4% paraformaldehyde, stored in 70% ethanol and later embedded in paraplast for histology. From the Hill collection kept at the Museum für Naturkunde in Berlin (Germany) we photographed histological slides of the cardiac region of all four specimens of developing Idiocranium russeli (ssc ZMB_EMB_AMP118–AMP121) in which this region was not too damaged. From the same collection we photographed histological slides of one adult Siphonops annulatus (ssc ZMB_EMB_AMP110). Using formalin-fixed and 70% ethanol-preserved specimens, we studied by dissection the gross morphology of the hearts of one specimen of each of Rhinatrema bivittatum (ssc MW2389), Ichthyophis tricolor (ssc MW1670), Scolecomorphus kirkii (ssc BMNH 2008.334), Herpele squalostoma (ssc BMNH 2008.334), Typhlonectes compressicauda (ssc MW7431, approximately 100 g), Typhlonectes natans (ssc MW5055, approximately 100 g) and the toad (Rhinella marina, 240 g). For further comparison we used the heart of a swordfish (donation from Kattegat Centret, Grenå, Denmark), a euthanised chimpanzee (donation from Givskud Zoo, Denmark) and published 3D models of hearts of a Geochelone tortoise (Jensen et al. 2014b), Burmese python, corn snake and ostrich (Jensen et al. 2013a).

Histology and immunohistochemistry

Excised hearts of Idiocranium were cut in 10-μm transverse or frontal sections. Myocardium was detected using immunohistochemistry, as previously described (Jensen et al. 2013a), using a rabbit polyclonal antibody to cardiac troponin I (cTnI) (Sc-15368, 0.5 μg mL−1, Santa Cruz) and visualised by a fluorescently labelled secondary goat-anti-rabbit antibody coupled to Alexa 488 (Invitrogen, dilution 1 : 250). Vessels were detected with a mouse monoclonal antibody (A2547, 1 : 250, Sigma) to smooth muscle actin (SMA) and visualised by a fluorescently labelled secondary goat-anti-mouse antibody coupled to Alexa 568 (Invitrogen, dilution 1 : 250). Nuclei were stained with DAPI (D9542, Sigma).

MRI scanning

The chimpanzee heart was MRI-scanned with a clinical Philips Achieva 1.5 T system (Philips Medical Systems, Amsterdam, the Netherlands). Data were acquired with a dedicated radiofrequency surface coil using high-resolution 3D gradient–echo sequence with the following parameters: field-of-view: 230 × 230 × 140 mm3, voxel size: 0.48 × 0.48 × 0.48 mm3, repetition time: 15.1 ms, echo time: 6.9 ms, excitation flip angle: 30°. The image series was exported as DICOM.

3D modelling

Sections of Idiocranium (MW9512–MW9513) were photographed, stacked, and aligned in Amira® v5.2, the DICOMs of the chimpanzee likewise, and then reconstructed as previously described (de Boer et al. 2011).

Results

Atrial septation in adult Idiocranium

The heart of Idiocranium, like the hearts of most caecilians, is very elongate. Unlike in most tetrapods, the oesophagus, trachea and associated arteries are found to the left of the heart, rather than dorsally, suggesting a general shift of the coelomic organs in the cardiac region. Within the heart, the interatrial septum is oriented from the right-sided cono-truncal region to the dorsal left and thus positioned obliquely and close to the frontal plane (Fig. 2A–C). It connects to the two leaflets of the atrioventricular valve, one leaflet on the left and the other on the right. The right atrium that harbours the sinuatrial orifice is essentially dorsal and the smaller left atrium that receives the pulmonary vein is largely ventral. The interatrial septum is largely myocardial with many pores, approximately 100. A left caudal part consists of connective tissue which is continuous with the connective tissue of both leaflets of the atrioventricular valve (Fig. 2A,E). Cranially the interatrial septum intermingles with numerous trabeculations and loses the appearance of a septum (Fig. 2D).

Fig. 2.

Fig. 2

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Anatomy of the atria of adult Idiocranium. (A). The interatrial septum (ias) is close to the left–right plane at the height of the sinuatrial orifice (sa). It contains a part of connective tissue (nuclear stain only). (A’) Interruptions in the Alcian blue stain of the section of (A) show multiple perforations of the interatrial septum (some are encircled). The pulmonary vein (pv) projects into the left atrial lumen and is continuous with the connective tissue part of the interatrial septum. (B) Trabeculations of the right atrial septum (ras) appear 90 μm cranial to (A). (C–C’) 260 μm cranial to (A) the right atrial septum spans the right atrium lumen from left to right and is thicker than the interatrial septum. (D) 650 μm cranial to (A), both atrial septa are hardly distinguishable from the atrial trabeculations. (E) Coronal (horizontal) section showing the close association of connective tissue part of the interatrial septum with the sinuatrial region, the pulmonary vein and the myocardial part of the interatrial septum. (F) Dorsal half of the reconstructed heart showing the leading edge of the right atrial septum (dotted red line) as a low arch associated with the sinuatrial valve (blue arrowhead) (myocardium is grey, connective tissue is yellow). (G) Left half of the reconstructed heart showing the atrial septum (reddish, cut edge indicated with black dots) and the right atrial septum (dotted red line), dividing ca. three-quarters of the right atrial lumen. avv, atrioventricular valve; cp, connective tissue part of the interatrial septum; cTnI, cardiac troponin I (detected myocardium); LA, left atrium; oft, outflow; R, right; RA, right atrium; SMA, smooth muscle actin; SP, sinus principale; ss, sinus septum; SS, sinus sinister; V, ventral; Ven, ventricle.

There is an additional septum within the right atrial cavity of Idiocranium, the right atrial septum (Fig. 2A–D). It is parallel to the interatrial septum and divides approximately three-quarters of the right atrium, creating a large blind compartment dorso-cranially (Fig. 2F,G). The right atrial septum is also myocardial and generally thicker than the interatrial septum (Fig. 2C). There are a few irregular communications through the septum, which appear distinct from the pores of the interatrial septum. Similar to the interatrial septum, the right atrial septum loses the appearance of a septum cranially as it intermingles with numerous trabeculations (Fig. 2D). The right atrial septum is continuous with the sinuatrial orifice (Fig. 2A,B,F,G) and resembles a prominent trabeculation associated with the sinuatrial valve of other vertebrate hearts (discussed further below).

Development of atrial septation in Idiocranium russeli

The hearts of all vertebrates form from a heart tube that loops and subsequently develops chambers by local ballooning (e.g. Jensen et al. 2013b). Consistent with this, in the youngest embryo of Idiocranium we studied (ssc ZMB_EMB_AMP118 characterised as ‘embryo including yolk’, Fig. 3A), the heart tube is looped such that the atrial cavity is approximately midline and the atrioventricular canal is left-sided. The still non-trabeculated ventricle is oriented from left to right and the myocardial outflow tract extends from the extreme right of the ventricle to the dorsal midline, where it is contiguous with the branchial arches (Fig. 3B). The atrial roof has a ridge of presumed mesenchyme. It is oriented with the neural folds, the notochord and the foregut (only lumen shown in the reconstruction) and is thus on the body midline (Fig. 3B). This mesenchymal ridge resembles the mesenchymal cap of the early septum primum of other tetrapods (Benninghoff, 1933; Jensen & Moorman, 2015). A later developmental stage (ssc ZMB_EMB_AMP119 characterised by ‘curled tip of tail behind gills’), here called mid-embryo (neural folds are fused to form the neural tube and the developing oesophagus and trachea are distinct but on the body midline), has atrial division by the interatrial septum, which is in the sagittal plane (Fig. 3C,D). Also, the two large atrioventricular cushions are dorsal and ventral respectively, not left and right as the leaflets they will become in the fully formed heart. A right atrial septum was not identified. In the most developed embryonic specimens (ssc ZMB_EMB_AMP120–121 characterised by ‘curled tip of tail behind gills’), here considered late embryo, the oesophagus, trachea and pulmonary vein were positioned on the left near the heart and the leaflets of the atrioventricular valve were positioned more left–right than dorso–ventral (Fig. 3E,F). The interatrial septum was oriented close to the dorso-ventral axis. Although trabeculations were found cranio-laterally in the right atrial cavity, a right atrial septum was not distinct (Fig. 3E,F). Development in Hypogeophis rostratus, reproduced from Schilling (1935), closely resembles that of Idiocranium (Fig. 3G,H). In summary, atrial septation in embryonic Idiocranium resembles that of other tetrapods and it appears that the right atrial septum and the atrial rotation are specialisations of late development.

Fig. 3.

Fig. 3

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Cardiac development in Indotyphlidae (A–F) Idiocranium russeli, (G–H) Hypogeophis rostratus. (A) Transverse section of a young embryo that includes looped heart tube with not yet trabeculated atrium (A) and ventricle (V). The roof of the atrium has a ridge (red arrowhead) on the body midline of presumed mesenchyme that may be the precursor of the atrial septum. (B) The looped heart tube has much cardiac jelly and mesenchyme (yellow) and the atrial septum precursor (red arrowhead) can be seen to align with other body midline structures (neural folds, notochord, foregut). (C) Near-transverse section of the much curved mid-embryo, showing the mesenchymal leading edge of the atrial septum oriented dorso-ventrally. (D). The oesophagus and trachea, with developing bronchi, remain on the body midline at this stage. (E) Transverse section of a late embryo showing the dorso-ventrally oriented atrial septum. (F) The lung vein is shifted to the left, possibly following the shift of the trachea and developing lungs (and oesophagus). (G–H) Wax models of the Hypogeophis rostratus heart from stages 35 and 45 (very little yolk left), unknown scale, modified from Schilling (1935). (G) is seen from the right, (H) shows the ventral surface. (G’–G’’) Sections of the wax model of (G) showing the early interatrial septum (red arrowheads) positioned on the dorso-ventral axis (G’ looking cranially, G’’ looking caudally). (H’–H’’) Sections of the wax model of H showing the early interatrial septum positioned at a skewed angle cranially (H’) but close to the dorso-ventral axis near the base of the ventricle (H’’). The right atrial septum (ras) is distinct, annotated in Schilling (1935) as ‘2. Scheidewand im rechten Vorhof’. avv, atrioventricular valve; C, chorda; E, oesophagus; LA, left atrium; lv, lung vein; N, notochord; Oft, outflow tract; RA, right atrium; SC, spinal cord; Tr, trachea;.

Atrial septation in caecilians

The interatrial septum in studied adult representatives of six additional families of caecilians is comparable to that of Idiocranium in its structure (disregarding details such as the size of septal perforations), although its orientation is variable. In Rhinatrema (Rhinatrematidae), representing the most plesiomorphic (ancestral) condition, the atrial cavity is divided by a single septum in the sagittal plane (Fig. 4B, see Wilkinson, 1996 for a detailed description). No right atrial septum is present. Ichthyophis (Ichthyophiidae) also has the interatrial septum in the sagittal plane. Ichthyophiids are reported as unique among caecilians in having partial external subdivision of the atria (Schilling, 1935; Wilkinson & Nussbaum, 1996). Surprisingly, the sulcus marking this external division is continued internally not by the interatrial septum, but by a dense mesh of myocardial trabeculations that forms a right atrial septum in the same sagittal plane as the interatrial septum (Fig. 4C,D). In Scolecomorphus (Scolecomorphidae), the interatrial septum is slightly off the sagittal plane ((Fig. 4E), with this oblique orientation representing the most plesiomorphic condition within the Teresomata (= all caecilians except Rhinatrematidae and Ichthyophiidae, Fig. 1). Laterally, trabeculations of the right atrial cavity form a sharp border to a medial part with very few trabeculations, representing a weakly developed yet sheet-like right atrial septum oriented in the dorso-ventral plane (Fig. 4E) as in Ichthyophiidae. In Herpele (Herpelidae) the interatrial septum and right atrial septum are both well-developed and oriented much closer to the frontal plane (Fig. 4F). A similar architecture is found in Typhlonectes compressicauda and T. natans (Typhlonectidae) (Fig. 4G). Based on histological sections rather than dissection, we found the base of the interatrial septum to be in the frontal plane and the two leaflets of the atrioventricular valve to be left and right, respectively, in Siphonops annulatus (Siphonopidae, not shown). The atria were so engorged with blood that we could not determine whether a right atrial septum was present but the study of Sawaya (1940; p. 233) mentions a ‘pequeno septo’ (small septum) of the right atrium.

Fig. 4.

Fig. 4

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Gross anatomy of atrial septation in caecilians. (A) Ventral aspect of the cardiac region of Rhinatrema (Rhinathrematidae). (B) The atrial cavity of Rhinatrema is partially divided by the dorso-ventrally oriented interatrial septum (ias) only. (C–D) In Ichthyophis (Ichthyophidae), in addition to the interatrial septum, a sulcus (arrowheads) partly compartmentalises the right atrial cavity and is continuous with a thick, trabeculate right atrial septum internally. (E) In Scolecomorphus (Scolecomorphidae) there is an oblique interatrial septum and a right atrial septum (ras) forming the border between a lateral region with trabeculation and a medial region with very little trabeculation. (F) Atrial septation in Herpele (Herpelidae) much resembles that of Scolecomorphus except it is more nearly in the frontal plane. (G) Dorsal half of the atrial cavity of Typhlonectes (Typhlonectidae), cut just dorsal of the interatrial septum, showing the division of the right atrial cavity by the right atrial septum (its leading edge is indicated with red dots). c, clot of blood behind the right atrial septum; LA, left atrium; Oft, outflow; RA, right atrium; V, ventricle.

The right atrial septum seems to be an apomorphic (derived) trait of the Ichthyophiidae and Teresomata, whereas the atrial rotation that renders the septa close to the frontal plane is seemingly confined to a subset of Teresomata (Fig. 5).

Fig. 5.

Fig. 5

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Atrial septation in amphibians. Names of the studied caecilian families are in black. In Anura and Caudata, the interatrial septum (ias) is always associated with the left-sided atrioventricular canal (avc) and oriented mostly in the dorso-ventral plane, but in some salamanders it may be oblique (e.g. Triton, Salamandra). Rhinatrematidae has the ancestral condition of one dorso-ventrally oriented atrial septum. Ichthyophidae has an external sulcus in addition to a right atrial septum (ras). Scolecomorphidae is likely to be the only Teresomata caecilian family without highly pronounced rotation of the atrial cavity and hence septation more nearly in the frontal plane. The phylogeny is based on San Mauro et al. (2014) and anatomical observations are partly from Benninghoff (1933), Schilling (1935), Davies & Francis (1941), Ramaswami (1944) and Wilkinson (1996). LA, left atrium; Oft, outflow tract; RA, right atrium.

Septum spurium and commissure of the sinuatrial valve in vertebrates

The right atrial septum appears to be an extremely well-developed septum spurium, a prominent trabeculation of the right side of the atrium that associates with the sinuatrial valve (Gallego et al. 1997; Jensen et al. 2014b). In mammals and birds with remodelling of the sinus venosus to the atria, the septum spurium will contribute to the terminal crest (or crista terminalis), the prominent trabeculation between the trabeculated atrial appendage and the smooth-walled and remodelled sinus venosus (Ho et al. 2002; Jensen et al. 2014a). We identified the septum spurium in the swordfish (teleost fish), marine toad (anuran amphibian), a Geochelone tortoise (reptile) and ostrich (bird), and in the chimpanzee in the remodelled form of the terminal crest (Fig. 6A). It spans the atrial roof like the right atrial septum but without forming a septum. In snakes, which are elongate like caecilians, the septum spurium (usually termed the suspensory ligament in reptiles; Jensen et al. 2014b) is no more than a prominent trabeculation and not a septum as seen in some caecilians (Fig. 6B). In some species, the septum spurium or the right atrial septum forms a circular structure by being continuous with trabeculations of the atrial floor that lead back to the region of the sinuatrial orifice (Fig. 6B, Typhlonectes and corn snake). These trabeculations are reminiscent of the trabeculations of the inferior isthmus of the human right atrium that fan out from the inferior part of the terminal crest (Ho et al. 2002; Sanchez-Quintana et al. 2002).

Fig. 6.

Fig. 6

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Septum spurium in vertebrates. (A). The septum spurium (dotted red line) associates with the sinuatrial valve (blue arrowhead) and is a common feature of the vertebrate heart, including the hearts of amphibians (black dotted line indicates the interatrial septum) (note that in the chimpanzee the sinus venosus is atrialised and the septum spurium is remodelled and contributes to the terminal crest, also marked by dotted red line). (B) In very elongate tetrapods, caecilians and snakes, the right atrial septum is only found in caecilians (exemplified by Typhlonectes natans). The septum spurium and the right atrial septum may be continuous with trabeculations that lead back to the region of the sinuatrial orifice (see T. natans and the corn snake) but such trabeculations are not easily traced. A, atrium; avv, atrioventricular valve; c, coagulated blood dorsal to the right atrial septum; LA, left atrium; LV; left ventricle; Oft, outflow tract; RA; right atrium; RV; right ventricle; SV, sinus venosus; V, ventricle.

Discussion

Atrial septation is always complete in amniotes and generally on the sagittal (dorso-ventral) axis in lungfishes and tetrapods (e.g. (Kardong, 2006). That the interatrial septum also is sagittal in caecilians of the families Rhinatrematidae and Ichthyophiidae strongly supports the inference that this is the ancestral orientation of the interatrial septum within the Gymnophiona. Atrial rotation and interatrial septation closer to the frontal plane appears to be a derived character of teresomatan caecilians, albeit with some variation in the degree of rotation (not as pronounced in Scolecomorphus). Atrial rotation may coincide with a left-ward shift of other visceral organs in the cardiac region during caecilian evolution. In support of this, our developmental studies on Idiocranium show that the late left-ward shift of the oesophagus and trachea coincides with a more left-sided position of the pulmonary vein and left–right positioning of the atrioventricular cushions. However, it is noteworthy that the interatrial septum is close to the sagittal axis in the late embryo stages and the frontal orientation of the atrial septa may not be tightly linked in time (ontogenetically or phylogenetically) to the left-ward shift of organs. It is an interesting open question why the organs shift in this way. This derived character is not obviously associated with changes in behaviour or habitat but might be associated with increased elongation of the body (members of the Rhinatrematidae and Ichthyophiidae are more thick-bodied and less attenuate than most other caecilians) and concomitant pressures on efficient visceral packing (Wilkinson, 1992). In the salamander Cryptobranchus, atrial septation is in the transverse plane concordant with a general rotation of the heart (Reese, 1906; Putnam & Parkerson, 1985) and this situation is likely analogous to the frontal septation reported here. In general, the caecilian right atrium is larger than the left, as is generally the case in amphibians and reptiles (Ramaswami, 1944; Jensen et al. 2014b).

Notes on heart development in the caecilian Typhlonectes compressicauda have been reported (Sammouri et al. 1990) but cardiac septation was not described. Our study, together with previous other brief reports (Schilling, 1935; Sawaya, 1940; Ramaswami, 1944) does not clarify when in development the adult position of the atrial septa is established. The latest stage investigated by Schilling, his stage 45 (Fig. 3H), was slightly older than our ‘late embryo’ stage and little yolk remains at this point (Schilling, 1935), but the atria were still not in the mature position. Thus, the final atrial rotation and positioning of the septa in the horizontal plane must be a late developmental event.

The interatrial septum of Idiocranium, and by extension of most caecilians, shares at least three characters with that of lungfishes and with the septum primum of embryonic amniotes. First, it has a basal part composed of connective tissue in between the atrioventricular valve and the pulmonary vein. This is like the pulmonary fold of lungfishes and the mesenchymal cap and dorsal mesenchymal protrusion, or spina vestibuli of His the elder, of embryonic amniotes (Briggs et al. 2012; Sylva et al. 2014; Jensen & Moorman, 2015). In mammals, the dorsal mesenchymal protrusion is necessary for complete atrial septum development and harbours the pulmonary vein primordium, whereas in caecilians, salamanders and lungfishes, we infer, it harbours the pulmonary vein only. Secondly, there is a gap between the atrial floor and the leading edge of the atrial septum (as the primary foramen in embryonic amniotes, e.g. Sylva et al. 2014). Lastly, the myocardial part of the atrial septum has multiple perforations (Klitgaard, 1978; Icardo et al. 2005b; Sylva et al. 2014). In Idiocranium, the perforations are much more numerous than in the septum primum, which has about 10 (for Idiocranium, see the histological sections in Fig. 2A’ and 2C’, where there are approximately 15 perforations to the interatrial septum in each) (Romanoff, 1960; Runciman et al. 1995; Macdonald et al. 2007; Anderson et al. 2014). In our opinion this is a large quantitative but not a qualitative difference and the perforations are lined by endocardium in Idiocranium and, for instance, marsupials alike (Röse, 1889; Runciman et al. 1995). The interatrial septum of many salamanders is also characterised by a free leading edge with connective tissue near the atrioventricular valves and a larger myocardial part with perforations (Bruner, 1900; Benninghoff, 1933; Davies & Francis, 1941; Putnam & Kelly, 1978; Putnam & Parkerson, 1978). Taken together, atrial septation appears evolutionarily conserved.

The right atrial septum appears to be a specialisation of the group including all caecilians except the Rhinatrematidae. Its absence in the Rhinatrematidae and in other elongate tetrapods such as snakes (Fig. 6), amphisbaenians (Francis, 1977), glass lizards (Acolat, 1943) and whales (Rowlatt, 1990) argues against its evolution being a direct consequence of body elongation. An obvious question is whether it contributes to separation of pulmonary and systemic venous blood streams. In Typhlonectes, which has a right atrial septum, Toews & MacIntyre (1978) found that systemic arterial blood had a higher oxygen tension than pulmonary arterial blood showing some capacity for intracardiac blood stream separation. However, to our knowledge this is all that is known of intracardiac shunting in caecilians. Intracardiac shunting is studied more in reptiles, also ectothermic tetrapods, and although the amphibian heart has more venues for shunting (atria, ventricle and conus) than the reptile heart (ventricle), the outcome is the same – reduced oxygen tension in the systemic arteries as compared with the pulmonary veins and increased oxygen tension in the pulmonary arteries as compared with the systemic veins (Hicks & Wang, 2012). In reptiles, long-term perturbations to the regulation of shunting seem to have little consequences for growth and performance (Hicks & Wang, 2012; Leite et al. 2013, 2014; Jensen et al. 2014b). We do not consider it very likely that the right atrial septum enhances fitness by improved separation of blood streams.

The anatomical substrate of the right atrial septum appears, at least in part, to be the septum spurium. The septum spurium is a particularly thick trabeculation of the (right) atrium of most vertebrates. In humans it associates with the cranial commissure of the sinuatrial valve seen in embryos and during developmental remodelling of the sinus venosus it contributes to an unknown extent to the terminal crest of fetal and formed hearts (Fig. 7) (some vertebrates have secondarily lost the sinuatrial valve and possibly the septum spurium as well; Jensen et al. 2014a). Ironically, (septum) spurium, like spurious, means false or fake (probably because the septum spurium is only transitorily septum-like in embryogenesis), but in the case of most caecilians this ‘fake’ septum is real. Cardiac septation is often associated with sulci, or clefts, on the epicardial surface (Rowlatt, 1990; Jensen et al. 2013b). It is therefore noteworthy that Ichthyophis of Ichthyophidae, the sister group of Teresomata, has an atrial sulcus associated with the right atrial septum. On morphological grounds, the right atrial septum is not like the septum secundum seen in placental mammal development. The septum secundum is a fold of the atrial roof between the interatrial septum and sinuatrial junction which, together with muscularised mesenchyme of the atrial floor, forms a large crescentic sheet that eventually covers the foramen ovale (Anderson et al. 2014).

Fig. 7.

Fig. 7

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The septum spurium in the placental mammal heart. (A) In hearts of embryonic humans the septum spurium (red arrow) is a prominent trabeculation associated with the cranial commissure of the left (L) and right (R) leaflets of the sinuatrial valve (ventral half of the heart of a Carnegie stage 14 embryo with myocardium in grey and mesenchyme in yellow; modified from Sizarov et al. (2011). (B) In fetal mouse hearts (day 17–18 in gestation), the septum spurium (red arrow) is a prominent trabeculation that has, in contrast to the terminal crest, trabeculations on both sides (adapted from Webb et al. (1996)). (C–E). In adult human (three specimens shown), the septum spurium (or sagittal bundle or taenia sagitallis) persists as a prominent trabeculation in the right atrial appendage, albeit with some variation in morphology (Loukas et al. 2008), and associates with the terminal crest (broken line) in the proximity of the superior caval vein (white asterisk) (adapted from Ho & Sanchez-Quintana, 2009). cs, coronary sinus; ICV, inferior caval vein; LA, left atrium; LV, left ventricle; OF, oval fossa; PF, primary foramen; RA, right atrium; RV, right ventricle; SP, septum primum.

Previous workers have disagreed over whether the interatrial septum of caecilians is complete or perforated and most have overlooked differences in its orientation and the presence of a right atrial septum. We conclude that the interatrial septum of caecilians may be complete or incomplete, may have perforations (Ramaswami, 1944; Wilkinson & Nussbaum, 1997) and shows multiple similarities with the interatrial septum of lungfishes and other tetrapods. All Teresomatans have atrial septation that is seemingly rotated from an ancestral sagittal orientation and may be either oblique or more nearly in the frontal plane. The derived, oblique or frontal orientation of the interatrial septum (which is mirrored in the orientation of the right atrial septum when present) probably follows a general shift and rotation of visceral organs late in development. A right atrial septum appears to be present in all caecilians except in Rhinatrematidae. The anatomical substrate of this derived structure appears to be the septum spurium, but its function is not known. Clearly, caecilian amphibians are a candidate for the vertebrate order with the most extreme variation in atrial septation.

Acknowledgments

Hearts were kindly donated to B.J. (swordfish, Kattegatcentret, Grenå, Denmark; Rhinella marina from Tobias Wang, Aarhus University, Denmark; Chimpanzee, Givskud Zoo, Denmark). The specimens of the Hill collection were kindly made available by Dr. Peter Giere of the Museum fÏr Naturkunde in Berlin (Germany). Michael Pedersen of Skejby Hospital, Aarhus University, Denmark, kindly scanned the heart of the chimpanzee. Antoon F. M. Moorman kindly critiqued an earlier version of the manuscript. B.J. was supported by The Danish Council for Independent Research I Natural Sciences. M.W. thanks Jeannot and Odette (Camp Patawa), Guy Tiego (Direction Régionale de l'Environnement, Cayenne), Myrian Virevaire (Direction de l'Environment de l'Aménagement et du Logement), Le Comitéé Scientifique Régional du Patrimonie Natureland Philippe Gaucher (Centre National de la Recherche Scientifique, Cayenne) for facilitating and Gabriela Bittencourt and David Gower for assistance in the collection of Rhinatrema bivittatum and Typhlonectes compressicauda in French Guiana. Cameroon fieldwork was funded by the Zoological Society of London (Erasmus Darwin Barlow grant) and Royal Zoological Society of Scotland. M.W. thanks the Cameroon Ministry of Forests and Wildlife for permissions and Thomas Doherty-Bone, Nono Gonwouo, David Gower, Marcel Kouete and Simon Loader for assistance in the field. We thank Gabriela Bittencourt for the photograph of Siphonops annulatus.

Author contributions

D.M.dB., M.W. and B.J. acquired and analysed the data and wrote the article.

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