The peculiar syrinx of Rhea americana ( Greater Rhea , Palaeognathae )

This work studied the skeletal and muscular syringeal anatomy of Greater Rhea (Rhea americana) throughout postnatal ontogeny, by using muscle staining and differential coloring of cartilage and bone techniques. Anatomical syrinx dissections on four adults (one female and three males) and eight unsexed chicks, were made. The type of the syrinx was tracheobronchial and it was entirely cartilaginous in chicks and in the adult female but showed a partially cartilaginous and osseous pessulus in male adults. A pair of intrinsic muscles were found and the extrinsic muscles were represented by the muscles sternotrachealis and tracheolateralis, and a broad dorsal medial muscular band. The syrinx of Greater Rhea was notable for having a more complex morphology than other Paleognathae birds. Future studies on how Rhea produces vocalizations will allow the comparison with other birds, and contribute to the understanding of the evolution of soundproduction mechanisms in birds.


Introduction
The Greater Rhea (Rhea americana) is the largest of South American birds, reaching 1.5 m in height and weigh ing 25 kg (Folch, 1992).Rhea is grouped with Ap teryx (kiwi), Casuarius (cassowary), Dromaius (emu) and Struthio (ostrich) in the Ratitae clade.This group together with Tinamidae (Tinamous) comprise the Pa laeognathae.These birds are monophyletic and a basal group with respect to Neognathae (e.g. livezey & zusi, 2007;Bourdon et al., 2009;PhilliPs et al., 2010;Johnston, 2011).Current studies on the Greater Rhea's anatomy are scarce (as well as in other Palaeognathae birds), despite their importance in the evolution and systematics of modern birds.
The syrinx is the organ responsible for producing vocalizations in birds and knowledge of its anatomy in Paleognathous birds is scarce and often outdated.Since the early 19 th century, studies on the anatomy of the syrinx have focused mainly on Neognathae birds (e.g.Müller, 1847;huxley, 1872;chaMBerlain et al., 1968;aMes, 1971;Warner, 1971Warner, , 1972;;GaBan-liMa & höFlinG, 2006;KaBaK et al., 2007;Miller et al., 2008).On the other hand, ForBes (1881) was the first to study the macro scopic anatomy of this organ on Ratites.Subsequently, Wun der lich (1884), Beddard (1898) and PycraFt (1900) corroborated these anatomical descriptions and pointed some minor anatomical variations (e.g.conformation and numbers of tracheal rings) but did not provide further details.The anatomy of the Palaeognathae syrinx would not be studied again until BeeBes (1925) works with the Variegated Tinamous (Crypturellus), and later on, a few others in the ostrich (Struthio;yildiz et al., 2003) and in Darwin's Nothura (Nothura; Garitanozavala, 2009).Beaver (1978) was the only researcher since the early 20 th century, to perform a brief and superficial anatomical description of the syrinx of chicks of the Greater Rhea.
The works of ForBes (1881), Wunderlich (1884), Bed dard (1898) and PycraFt (1900) state that the syrinx of the Greater Rhea has intrinsic muscles and a welldeveloped tympanum unlike that of other Ratites.Also, Wun derlich (1884) was the first to mention the cartilaginous nature of the pessulus.However, due to methodological limitations at the time, these studies did not provide detailed anatomical descriptions, nor age or sex of the individuals studied.Also, the illustrations only show two macroscopical syrinx drawings in dorsal and ventral aspects.
Studies on the comparative morphology of the syrinx contribute to understand the mechanisms of sound production (e.g.Goller & larsen, 1997;larsen & Goller, 1999larsen & Goller, , 2002) ) and have been used to establish phylogenetic relationships in systematic studies (e.g. aMes, 1971;livezey, 1986;PruM, 1992;GriFFiths, 1994;GaBan-liMa & höFlinG, 2006;ziMMer et al., 2008;MandiWana et al., 2011).The aim of this study was to describe the anatomy of the syrinx in Rhea americana throughout its ontogeny, by using muscle staining (BocK & shear, 1972) and differential colouring of cartilage and bone (cannel, 1988).This approach to the study of the anatomy of the syrinx of a Ratite bird would be useful to carry out further studies on its comparative and functional anatomy.

Materials and Methods
Anatomical dissections were performed on 12 specimens of Rhea americana at several ages: four adults (1 female and 3 males of two years old), four 3-month-old unsexed chicks, and four 1-month-old unsexed chicks.The birds were obtained from various commercial farms located in Buenos Aires province (Argentina) and reared in accordance with Argentinean regulations for Greater Rhea farming.Birds were sacrificed by cervical dislocation or electrical stunning, and their syringes were first observed in situ and then they were carefully removed.They were fixed in 10 % formaldehyde for 48 hours and then preserved in 70 % ethanol.An iodine solution that selectively stains muscular tissue with a reddish brown color (BocK & shear, 1972) was used to observe the muscles in preserved syringes.Lastly, the syringes were stained using a standard differential coloring technique for cartilage and bone (cannels, 1988), where cartilage tissues are stained blue (alcian blue) and ossified tissues are stained red (alizarin red).The anatomical nomenclature followed throughout corresponds to that proposed by BauMel et al. (1993).Photographs were taken with a Nikon D-40 digital camera.

Skeletal elements of the adult syrinx
The syrinx of Greater Rhea was of the tracheobronchial type and it was composed of cartilages, namely (cart.)tracheosyringeales (T) and cart.bronchosyringeales (B).The pessulus was a thin bar, fused with the tympanum (figs.2a,b & 3).In females, the pessulus was entirely cartilaginous (figs. 2a, b).On the other hand, in males, it was cartilaginous on its dorsal half, whereas it was osseous on its ventral half, forming an osseous plate (figs.2c, d).The membrana tympaniformis lateralis (fig. 1) was located between the last cart.tracheosyringeal (T4) and the first cart.bronchosyringeal (B1).It was partially covered by the pair of intrinsic muscles (fig.1; see below).The membrana tympaniformis medialis (figs. 1 & 3a) was suspended between the free ends of the cart.bronchosyringeales B1 and B2, and extended, making contact with the pessulus (fig.3a).This membrane formed a pair of intrusions into the lumen of the syrinx (fig.3b).Finally, the ligamentum interbronchiale connected the left and right bronchii (fig. 1) and between this ligament and the pessulus the foramen interbronchiale could be observed (fig.1).

The syrinx of chicks
In chicks, the syringes were cartilaginous and presented similar anatomy regardless of age (figs.2e, f).Unlike adults, the cart.bronchosyringeal B1 was not fused with the cart.tracheosyringeal T4 in its ventral aspect (figs.2e, f).Other differences were noted in the first cart.tracheosyringeales: the fusion (or joint zones) between cart.T1 -T3 was variable among the chicks considered in this study, as well as the presence of a partial bifurcation (divergence zones) of these cartilagones (figs.2e, f).

Syrinx musculature
The musculature of the syrinx consisted of extrinsic and intrinsic muscles.The extrinsic musculature was represented by paired muscles (mm.), namely sternotrachealis and tracheolateralis (figs.4a -c), and by a broad muscular band located on the mid-dorsal region of the trachea (figs. 4b, e).The muscle (m.) sternotrachealis was thin and delicate; it originated in the internal surface of the sternum and inserted on each side of the trachea, cranially to the tympanum (fig.4a).The m. tracheolateralis (figs.4b,c), was located on the lateral surface of the trachea; it was thin and weak, originating on the lateral surface of the larynx and inserting near to the cranial attachment of the intrinsic muscles (fig.4c).The broad muscular band (figs.4b, e) was thin and firmly attached to the dorsal surface of the trachea from near the larynx to the tympanum (at the level of the cart.T1 & T2).A single pair of intrinsic muscles were observed (figs. 4a, d).They consisted on wide and thin muscular bands with an oblique path extending from the dorsal region of the cart.tracheales (immediately preceding the tympanum) to the ventral surface of the first three cart.bronchosyringeales.In unpreserved specimens, all muscles were thin and delicate and presented a pink pale color (figs.4a & 1).No differences were found in muscle arrangement through ages.

Discussion
The syrinx of the Greater Rhea corresponds to the tracheobronchial type, as in the remaining Palaeognathae birds (ForBes, 1881;yildiz et al., 2003;Garitano-zavala, 2009) and most Neognathae birds (Beddard, 1898;KinG, 1989;BauMel et al., 1993).But, the Greater Rhea syrinx was notable for having a well-developed tympanum, and for the presence of a pair of intrinsic muscles.In the rest of the Paleognathae birds, there are no intrinsic muscles and the tympanum has often been described as simple due to the presence of a lower degree of fusion between the components and the presence of a pessulus of connective tissue (ForBes, 1881;Wunderlich, 1884;yilidz et al., 2003;Garitano-zavala, 2009).ForBes (1881) described the presence of a "vocal cord" inside the syrinx of Rhea americana (p.240).It is possible that this author called "vocal cord" the two intrusions of the membranae tympaniformes mediales into the lumen of the syrinx (see fig. 3a).These intrusions are noticeable when the syrinx is fixed and preserved, whereas in, unpreserved specimens this is not as evident.In regard to the extrinsic muscles, the results of this study showed some differences with respect to information given by previous authors (i.e.: ForBes, 1881, Wunderlich, 1884;Beddard,1898;PycraFt, 1900).Initially, these authors described the presence of a single pair of extrinsic muscles which were identified as the "lateral tracheal muscle" (ForBes, 1881 p. 240), without giving further details on their origin and extension.In our work, we identified the two typical pairs of extrinsic muscles of the syrinx (mm.sternotracheales and tracheolaterales).When comparing it with other Palaeognathae birds, ForBes (1881) found that the ostrich syrinx had no intrinsic or extrinsic muscles, but Wunderlich (1884), PycraFt (1900) and yilidz et al. (2003) described the presence of the m.sternotrachealis.Regarding the remain genera (Casuarius, Dromaius and Apteryx), these authors described the presence of the two typical pairs of extrinsecal muscles.These disparities indicate that variations in the musculature (e.g.presence or absence of a muscle) is a common trait in birds (BerGer, 1956;BerMan et al., 1990).
Lastly, concerning the medium-dorsal muscular band present in the Greater Rhea, ForBes (1881) called it "fibrous band" and mentioned its presence also in the Cassowary.After a review of the available bibliography, we could not find a similar muscle in other birds.
The presence of intrinsic muscles in the syrinx of the Greater Rhea is interesting.They are a distinctive feature of Passerine birds (especially the Oscines songbirds), that have at least four pairs that contribute to the control of a wide variety of vocalizations (suthers, 2001;larsen & Goller, 2002).Some groups of non-Passeriformes birds also have intrinsic muscles, although in lesser numbers, namely hummingbirds (Trochiliformes, Müller, 1847;Gaunt, 1983), parrots (Psittaciformes, Gaunt & Gaunt, 1985;GaBan-liMa & höFlinG, 2006) and the oilbird  hector, 1985).These birds do not have a large repertoire of vocalizations, but they can imitate sounds (e.g.parrots) and are capable of vocal learning (e.g.hummingbirds and parrots) (Gaunt, 1983;Gaunt & Gaunt, 1985;suthers, 2001).Moreover, in the echolocating oilbird, the intrinsic muscles have evolved for the production of sonar clicks (suthers & hector, 1985).The role that intrinsic muscles perform in the functioning of the syrinx of the Greater Rhea is still unknown, however, is interesting to note that adults of R. americana produce sounds like "hisses" and during breeding season, male adults make a deep-toned "grunt" (termed booming call), that can be heard at great distances (raiKoW, 1969;BruninG, 1974;Beaver, 1978;Folch, 1992;codenotti & alvarez, 2001;davies, 2002).In contrast, Beaver (1978) found that young birds have a wide repertoire of sounds (consisting of about five types of vocalizations), that impoverishes and disappears as the chicks grow.This author related this vocal modification with the more developed membranae tympaniformes mediales in chicks than in adults.Nonetheless, he did not perform measurements on this membrane to corroborate this statement and the published figures are unclear (see Beaver, 1978 fig. 3, p. 387), without scale and taken in different views.In our study no macroscopic differences were found in the membranae tympaniformes mediales when comparing chicks to adults.We also believe that the degree of intrusion of the membrane could be a fixation artifact.Therefore, we conclude that the impoverishment of richness of sounds found by Beaver (1978) should be studied from other perspectives.
The syrinx of the Greater Rhea was previously described as being completely cartilaginous (ForBes, 1881;Wunderlich, 1884;PycraFt, 1900), but the differential staining techniques incorporated in this study showed the presence of a pessulus in the male adult formed by osseous and cartilaginous tissues.A similar condition (yet of unknown significance) was found in the male of the Tufted Duck, Aythya fuligula (Warner, 1971).Sexual dimorphism in syrinx anatomy has been found in other birds (Miller et al., 2008), varying from the presence of larger syringes in males (e.g. in the collared dove, Streptopelia decaoto, BallintiJn & cate, 1997), to the presence of specialized structures such as the syringeal bulla in males of Anatidae (e.g.FranK et al., 2007;Warner, 1971), to differences in structures like labia and cartilaginous rings (e.g. in the Zebra Finch, Taeniopygia guttata; riede et al., 2010).Also, differences in vocalizations between male and female could be associated with sexual dimorphism in syrinx anatomy (BallintiJn & cate, 1997;Miller et al., 2008;riede et al., 2010;Warner, 1971), but in the Greater Rhea this topic still remains to be explored.
In conclusion, the present study points to the complexity in the morphology of the syrinx of Rhea ameri cana and the more attention it deserves.Future studies on air flow and air sac pressure, intrinsic and extrinsic muscles electromyography and endoscopic filming of the syrinx during the generation of sounds are needed to complement these findings.Such information will allow to compare if the mechanisms of sound production of Palaeognathae differs from those known for Neognathae birds, and eventually shed new light on the evolution of this feature in birds.

Fig. 4 .
Fig. 4. Musculature of the syrinx.(a) In situ ventral view of the syrinx showing the m.sternotrachealis (mst) and the right intrinsic muscle (im) (specimen of three months old); (b) and (c) Proximal and caudal left view respectively of the syrinx showing the origin and insertion of m. tracheolateralis (mtl) (specimen of one month old); (d) Left lateral view showing the left intrinsic muscle (im) (specimen of three month old); (e) Dorsal view showing the medial muscular band (mb) (specimen of three month old), (b -e): specimens stained with iodine solution, bp: bronchus primarius, la: larynx, st: sternum, to: tongue, tr: trachea, ty: tympanum.Arrow in figures b and c indicates dorsal surface.