Abstract

Chondrichthyes, a monophyletic group comprising sharks, batoids, and chimaeras, includes approximately 1200 valid species and displays remarkable morphological diversity, both internal and external. Among soft tissues, the heart and its associated structures in cartilaginous fishes remain understudied, with most research focusing on circulatory physiology rather than comparative anatomy. This study provides a detailed description of the morphological variation of the afferent hyoid and branchial arteries across 13 of the 14 recognized orders of Chondrichthyes. A total of 16 anatomical characters associated with this complex were analyzed, seven were previously described, with two being redefined, and nine others are proposed as new. All characters were tested cladistically in order to explore evolutionary patterns. Our findings were able to expand the current knowledge of the morphological diversity of afferent branchial arteries in Chondrichthyes and provide new insights into the evolution of cardiovascular structures within this lineage.

Anatomy, circulatory system, Elasmobranchii, Holocephali, morphology

Cartilaginous fishes form a separate vertebrate class composed of sharks, batoids, and chimaeras, comprising approximately 1200 valid species (Weigmann et al. 2024; Fricke et al. 2025). The monophyly of Chondrichthyes and its major groups, Elasmobranchii (sharks and rays) and Holocephali (chimaeras), has been recovered by several morphological and molecular studies (e.g., Compagno 1977; Shirai 1996; Douady et al. 2003; McEachran and Aschliman 2004; Naylor et al. 2012a). However, the phylogenetic relationships among chondrichthyan orders and families remain unresolved. For instance, different placements have been proposed among batoid orders, with Torpediniformes, Rajiformes or Pristiformes as the sister group to all other living batoids (Compagno 1977; Shirai 1996; McEachran et al. 1996; Aschliman et al. 2012a, 2012b; Naylor et al. 2012a; Pavan-Kumar et al. 2014; Naylor 2025). Other unsolved issues concern the monophyly of the order Rhinopristiformes, (sawfishes and guitarfishes) and the sister-taxa relationship between Rhinochimaeridae and Chimaeridae (Didier 1995; Naylor et al. 2012b).

Phylogenetic research carried out through the analysis of morphological characters is more commonly based on skeletal characters (Shirai 1996; Aschliman et al. 2012a; Da Silva and De Carvalho 2015; Soares and De Carvalho 2020; Villalobos-Segura et al. 2022) with soft tissue characters being less commonly used (Northcutt 1989; Soares and De Carvalho 2013; Soares and Toledo-Piza 2021; Soares 2024). Previous studies of soft tissues have shown that the heart and its arteries show a significant amount of variation that could have phylogenetic constraints (Muñoz-Chápuli et al. 1994; Soares and Toledo-Piza 2021). For instance, Soares and Toledo-Piza (2021) described the morphological variation in afferent branchial arteries of several batoids and proposed seven morphological characters related to these arteries, including a synapomorphic condition observed for Rajiformes. Another example is Muñoz-Chápuli et al. (1994), who proposed a derived condition based on the arrangement of coronary arteries shared between Rajiformes and Myliobatiformes.

However, previous studies have focused only on smaller groupings within Chondrichthyes, such as Soares and Toledo-Piza (2021) on Batoidea, Miyake (1988) on myliobatiforms, and Allis (1912a, 1912b, 1912c) on Amblyraja radiata (Donovan, 1808), Heptranchias perlo (Bonnaterre, 1788) and Hydrolagus colliei (Lay & Bennett, 1839). Therefore, it is very likely that there is still morphological variation in the heart structures of Chondrichthyes awaiting discovery. Therefore, the present study provides a detailed examination of the morphological variation in afferent branchial arteries across Chondrichthyes, proposing and testing characters within a phylogenetic framework. By doing so, we seek to deepen our understanding of the branching patterns of these arteries and their phylogenetic significance within the class Chondrichthyes.

The specimens examined were obtained from museum collections and the Laboratory of Chondrichthyes of the Universidade Federal do Rio de Janeiro (LC-UFRJ). A total of 101 specimens were examined, belonging to 95 species, 44 families, and 13 of the 14 extant chondrichthyan orders. Two specimens were examined as comparative material and outgroups, the teleost Caranx hippos (Linnaeus, 1766) (Osteichthyes) and the hagfish Eptatretus burgeri (Girard, 1855) (Myxiniformes). Classification of elasmobranch orders follows Weigmann et al. (2024). Although we aimed to have at least one specimen from each order, given the unavailability of Echinorhinus, the only genus from the order Echinorhiniformes, for destructive analysis, it was not possible to include it.

Specimens examined are deposited in the following institutions: CAS, California Academy of Sciences, San Francisco; LC-UFRJ, Laboratory of Chondrichthyes, Rio de Janeiro; MCZ, Museum of Comparative Zoology, Cambridge; MNRJ, Museu Nacional, Rio de Janeiro; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo; UERJ, Universidade do Estado do Rio de Janeiro, Rio de Janeiro. The abbreviations TL and DW used throughout the text refers to total length and disc width, respectively. A list of the material examined is provided below.

Carcharhiniformes . Atelomycteridae: Atelomycterus fasciatus Compagno & Stevens, 1993, MZUSP 118095, female, 363 mm TL (Australia). Carcharhinidae: Carcharhinus brevipinna (Müller & Henle, 1839), LC-UFRJ 012, female, 950 mm TL (Rio de Janeiro, Brazil); C. glaucus (Linnaeus, 1758), UERJ 1670.3, female, 370 mm TL (no locality data); C. obscurus (Lesueur, 1818), LC-UFRJ 108, male, no TL data, (Maricá, Rio de Janeiro, Brazil); C. limbatus (Valenciennes, 1839), LC-UFRJ 031, no sex or TL data, (Grumari, Rio de Janeiro, Brazil); Negaprion brevirostris (Poey, 1868), MNRJ 16588, male, 625 mm TL (Atol das Rocas, Rio Grande do Norte, Brazil); Rhizoprionodon lalandii (Valenciennes, 1839), LC 063, no sex or TL data (Copacabana, Rio de Janeiro, Brazil); R. porosus (Poey, 1861), LC-UFRJ 081, female, no TL data (Copacabana, Rio de Janeiro, Brazil); Triaenodon obesus (Rüppell, 1837), LC-UFRJ 138, female, 430 mm TL (no locality data). Galeocerdonidae: Galeocerdo cuvier (Péron & Lesueur, 1822), MZUSP uncatalogued, female, 895 mm TL (São Paulo, Brazil). Pentanchidae: Apristurus australis Sato, Nakaya & Yorozu, 2008, MZUSP 118094, female, 493 mm TL (Australia, 33°49'S, 151°57'E); Galeus arae (Nichols, 1927), MCZ 40169, male, 260 mm TL (Chile). Proscylliidae: Eridacnis barbouri (Bigelow & Schroeder, 1944), MCZ 36135, female, 270mm TL (Cuba, 22°25'58.25''N, 78°45'10.56''W). Scyliorhinidae: Scyliorhinus haeckelii (Miranda Ribeiro, 1907), MNRJ 33570, male, 415 mm TL (Brazil). Sphyrnidae: Sphyrna zygaena (Linnaeus, 1758), LC-UFRJ 058, no sex or TL data (Copacabana, Rio de Janeiro, Brazil). Triakidae: Mustelus canis (Mitchill, 1815), MNRJ 18723, female, 520 mm TL (Rio Grande do Sul, Brazil).

Chimaeriformes . Callorhinchidae: Callorhinchus callorynchus (Linnaeus, 1758), MNRJ 3715, male, 540 mm TL (Rio de Janeiro, Brazil). Chimaeridae: Hydrolagus matallanasi Soto & Vooren, 2004, MNRJ 42861, female, 470 mm TL (14°28'23''S, 38°52'24''W). Rhinochimaeridae: Rhinochimaera atlantica Holt & Byrne, 1909, MNRJ 38971, male, 520 mm TL (no locality data).

Heterodontiformes . Heterodontidae: Heterodontus francisci (Girard, 1855), MCZ 155798, female, 380 mm TL (no locality data).

Hexanchiformes . Hexanchidae: Heptranchias perlo, MNRJ 30211, female, 670mm TL (Brazil, 14°22.3'S, 38°40.12'W); Notorynchus cepedianus (Péron, 1807), LC-UFRJ 128, no sex or TL data (Copacabana, Rio de Janeiro, Brazil).

Lamniformes . Lamnidae: Isurus oxyrinchus Rafinesque, 1810, LC-UFRJ 122, no sex or TL data (Copacabana, Rio de Janeiro, Brazil). Mitsukurinidae: Mitsukurina owstoni Jordan, 1898, MZUSP 130949, male, 108 mm TL (Asia). Odontaspididae: Carcharias taurus Rafinesque, 1810, MNRJ 3899, female, 540 mm TL (Rio de Janeiro, Brazil). Pseudocarchariidae: Pseudocarcharias kamoharai (Matsubara, 1936), MZUSP uncatalogued, female, no TL data (no locality data).

Myliobatiformes . Aetobatidae: Aetobatus narinari (Euphrasen, 1790), LC-UFRJ, no sex or TL data (Rio de Janeiro, Brazil). Dasyatidae: Brevitrygon manjajiae Last, Weigmann & Naylor, 2023, USNM 222624, female, 520 mm TL, 220 mm DW (India); Brevitrygon walga (Müller & Henle, 1841), USNM 432188, male, 510 mm TL (India); Dasyatis hypostigma Santos & Carvalho, 2004, LC-UFRJ 123, female, 430 mm DW (Copacabana, Rio de Janeiro, Brazil); Hypanus guttatus (Bloch & Schneider, 1801), LC-UFRJ 124, male, 1060 mm TL, 390 mm DW (Copacabana, Rio de Janeiro, Brazil); H. marianae (Gomes, Rosa & Gadig, 2000), MZUSP 52885, female, 427 mm TL (Abrolhos, Bahia, Brazil); Neotrygon leylandi (Last, 1987), LC-UFRJ 194, female, 380 mm TL, 220 mm DW (Nickol Bay, Australia); Pateobatis bleekeri (Blyth, 1860), USNM 222606 male, 300 mm TL, 120 mm DW (Bangladesh, India). Gymnuridae: Gymnura altavela (Linnaeus, 1758), MNRJ 41122, male, 290 mm TL, 460 mm DW (Rio de Janeiro, Brazil, 22°41'56''S, 43°6'30''W); G. micrura (Bloch & Schneider, 1801), MZUSP 122987, female, 255 mm TL (Caiçara do Norte Beach, Rio Grande do Norte, Brazil, 5°3'45''S, 36°3'45''W). Hexatrygonidae: Hexatrygon bickelli Heemstra & Smith, 1980, MZUSP 130502, male, 400 mm TL, 350 mm DW (Taiwan). Myliobatidae: Aetomylaeus bovinus (Geoffroy Saint-Hilaire, 1817), CAS 34943, female, 560 mm TL, 345 mm DW (no locality data); Myliobatis freminvillei Lesueur, 1824, MZUSP 9927, female, 672 mm TL (Rio Grande do Sul, Brazil, 29°23'S, 49°16'W). Mobulidae: Mobula hypostoma (Bancroft, 1831), MZUSP 59291, male, 751 mm TL (Guarujá, São Paulo, Brazil). Plesiobatidae: Plesiobatis daviesi (Wallace, 1967), MZUSP 130509, male, 460 mm TL, 420 mm DW (Asia). Potamotrygonidae: Potamotrygon motoro (Müller & Henle, 1841), MZUSP 82468, female, 420 mm TL (Tapajós River, Pará, Brazil, 2°24'46''S, 54°53'30''W). Rhinopteridae: Rhinoptera bonasus (Mitchill, 1815), MZUSP 72932, male, 568 mm TL (Barequeçaba Beach, São Paulo, Brazil). Urolophidae: Urolophus viridis McCulloch, 1916, LC-UFRJ 195, female, 380 mm TL, 240 mm DW (Merimbula, Wales); Trygonoptera galba Last & Yearsley, 2008, LC-UFRJ 193, female, 480 mm TL, 250 mm DW (Rottwest Island, Australia). Urotrygonidae: Urobatis halleri (Cooper, 1863), USNM 181332, male, 315 mm TL (Isla Santa Margarita, Baja California, Mexico); Urotrygon microphthalmum Delsman, 1941, MCZ 4032, male, 180 mm TL, 85 mm DW (Brazil, 2°34'N, 49°18'W); Urobatis jamaicensis (Cuvier, 1816), USNM 38642, female, 250 mm TL, 125 mm DW (Colombia).

Orectolobiformes . Ginglymostomatidae: Ginglymostoma cirratum (Bonnaterre, 1788), MNRJ 7962, female, 670 mm TL (Recife, Pernambuco, Brazil). Hemiscyllidae: Chiloscyllium plagiosum (Bennett, 1830), MCZ S-50, female, 480 mm TL (Hong Kong, China).

Pristiophoriformes . Pristiophoridae: Pristiophorus japonicus Günther, 1870, MZUSP uncatalogued, female, no TL data (Japan).

Rajiformes . Anachantobatidae: Schroederobatis americana (Bigelow & Schroeder, 1962), MCZ 42953, female, 306 mm TL (French Guiana). Arhynchobatidae: Atlantoraja castelnaui (Miranda Ribeiro, 1907), MNRJ 32319, male, 216 mm TL (Rio de Janeiro, Brazil); A. cyclophora (Regan, 1903), MZUSP 117184, female, 583 mm TL (Itajaí, Santa Catarina, 26°41'S, 46°42'W); Notoraja azurea McEachran & Last, 2008, MZUSP 125619, female, 466 mm TL (New Zealand); Psammobatis extenta (Garman, 1913), MNRJ 32468, female, 370 mm TL (Rio de Janeiro, Brazil); Rioraja agassizii (Müller & Henle, 1841), MNRJ 50512, female, 505 mm TL (Rio de Janeiro, Brazil, 23°11'30''S, 42°58'30''W); Sympterygia acuta Garman, 1877, MCP 4745, male, 525 mm TL (Rio Grande do Sul, Brazil, 32°14'S, 52°6'60''W), S. bonapartii Müller & Henle, 1841, MZUSP 11781, female, 416 mm TL (Santa Catarina, Brazil, 27°38'52''S, 48°20'16''W). Gurgesiellidae: Cruriraja rugosa Bigelow & Schroeder, 1958, MCZ 41970, female, 318 mm TL (Caribbean Sea); Fenestraja plutonia (Garman, 1881), MCZ 51820, female, 213 mm TL (Florida, United States); Gurgesiella atlantica (Bigelow & Schroeder, 1962), MNRJ 9535, female, 370 mm TL (Suriname, 7°15'N, 53°21'W). Rajidae: Amblyraja radiata, MCZ 52692, male, 223 mm TL (Gulf of Maine, Massachusetts, United States); Breviraja nigriventralis McEachran & Matheson, 1985, MCZ 42948, female, 269 mm TL (southwestern Atlantic); Dactylobatus clarkii (Bigelow & Schroeder, 1958), MCZ 51810, male, 369 mm TL (Magdalena, Colombia); Dipturus sp., UERJ E505, male, 426 mm TL (no locality data); Leucoraja garmani (Whitley, 1939), MCZ 37162, female, 317 mm TL (Gay Head, Massachusetts, United States); Malacoraja senta (Garman, 1885), MCZ 37091, male, 284 mm TL (New England, United States); Raja miraletus, USNM 193737, male, 408 mm TL (Liberia); Rajella purpuriventralis (Bigelow & Schroeder, 1962), UERJ uncatalogued, female, 390 mm TL (São Paulo, Brazil); Rostroraja equatorialis (Jordan & Bollman, 1890), USNM 222220, female, 339 mm TL (Colombia); Zearaja chilensis (Jordan & Bollman, 1890), MCP 3756, male, 278 mm TL (Uruguay);

Rhinopristiformes . Glaucostegidae: Glaucostegus granulatus (Cuvier, 1829), USNM 149733, male, 339 mm TL (India). Pristidae: Pristis pectinata Latham, 1794, MPEG 2324, female, no TL data (Pará, Brazil). Rhinidae: Rhynchobatus australiae Whitley, 1939, LC-UFRJ uncatalogued, female, 430 mm TL, 160 mm DW (Australia); R. djiddensis (Forsskål, 1775), CAS 247073, female, 390 mm TL (Sri Lanka); R. palpebratus Compagno & Last, 2008, MZUSP 125618, male, 576 mm TL (Queensland, Australia, 12°24'3''S, 12°25'9''E). Rhinobatidae: Pseudobatos horkelii (Müller & Henle, 1841), MNRJ 51463, female, 652 mm TL (Rio de Janeiro, Brazil, 21°36'58''S, 41°2'55''W); Rhinobatos sainsbury Last, 2004, MZUSP, female, 550 mm TL (Dampier Archipelago, Austrália). Trygonorrhinidae: Zapteryx brevirostris (Müller & Henle, 1841), MZUSP 117272, 455 mm TL (Ubatuba, São Paulo, Brazil).

Squaliformes . Etmopteridae: Etmopterus bigelowi Shirai & Tachikawa, 1993, MNRJ 0517, female, 630 mm TL (no locality data). Somniosidae: Centroscymnus owstonii Garman, 1906, MNRJ 40549, female, 399 mm TL (Espírito Santo, Brazil, 21°46'34''S, 39°53'22''W). Squalidae: Squalus albicaudus Viana, Carvalho & Gomes, 2016, USP uncatalogued, 4 specimens (no locality data).

Squatiniformes . Squatinidae: Squatina guggenheim Marini, 1936, MZUSP 42851, female, 532 mm TL (Brazil, 28°43'S, 48°20'W); Squatina occulta Vooren & da Silva, 1991, MNRJ 33235, male, 350 mm TL (Rio de Janeiro, Brazil, 23°4'45''S, 43°8'92''W).

Torpediniformes . Narcinidae: Benthobatis kreffti Rincón, Stehmann & Vooren, 2001, MZUSP 86555, female, 264 mm TL (Brazil, 25°46'11''S, 45°11'49''W); Discopyge tschudii Heckel, 1846, MZUSP 72780, female, 291 mm TL (Uruguay, 35°18'S, 54°13'W); Narcine brasiliensis (Olfers, 1831), MZUSP 72799, male, 263 mm TL (Peruíbe, São Paulo, Brazil). Narkidae: Heteronarce sp., CAS 58351, female, 232 mm TL (Somalia); Temera hardwickii Gray, 1831, CAS 35736, 122 mm TL (Singapore). Platyrhinidae: Platyrhinoidis triseriata (Jordan & Gilbert, 1880), CAS 59621, male, 383 mm TL, CAS 31248, female, 313 mm TL (United States). Torpedinidae: Tetronarce puelcha (Lahille, 1926), MZUSP 86769, male, 296 mm TL (Brazil, 23°44'17''S, 42°12'W).

Comparative material. Carangiformes. Carangidae: Caranx hippos, LC-UFRJ 51, no sex data, 195 mm TL (Itacaré, Bahia, Brazil). Myxiniformes. Myxinidae: Eptatretus burgeri, LC-UFRJ 024, no sex data, 565 mm TL (no locality data).

Descriptions of the heart and afferent branchial arteries were based on specimens previously fixed with formaldehyde and preserved in 70% ethanol. Manual dissections were carried out with the aid of forceps, scalpels, excavators, and fine-tipped scissors. Two incisions were made on the ventral surface of specimens, one horizontal, just anterior to the base of both pectoral fins, and one vertical, running from the pectoral girdle to the mouth symphysis. With that, we were able to remove the skin and hypobranchial muscles to uncover the heart and afferent branchial arteries, which were removed from the pericardial cavity and stored in 70% ethanol. Photographs of the heart and its arteries were taken with a Canon EOS 5D Mark III and edited with the aid of Adobe Photoshop CS6.

The terminology used to describe the afferent branchial arteries follows Soares and Toledo-Piza (2021). A total of nine characters were proposed herein, in addition to seven previously described by Soares and Toledo-Piza (2021), and all 16 characters were subsequently added to more comprehensive morphological matrices, such as that of Shirai (1996) and Aschliman et al. (2012a). Afterward, all data matrices were analyzed under maximum parsimony with the program TNTv1.5 (Goloboff and Catalano 2016) using the Traditional search method with 1 random seed and 100 replicates as the number of additional sequences for Wagner trees, tree bisection–reconnection (TBR) branch swapping, and retained trees limit set to 5000. The ‘map common synapomorphies' tool, was used to map the characters states, by plotting them into the generated consensus tree and identifying the shared character states between the branches. Additionally, the relative Bremer support value of each terminal taxon (Bremer 1994; Goloboff and Farris 2008) was calculated, retaining suboptimal trees with twelve steps. Additionally, the consistency (IC) and retention (IR) indices and frequency differences (GC) were also calculated. The resulting trees were edited in FigTree 1.3.1 (Rambaut 2010) and Adobe Photoshop CS6.

The branching pattern of afferent hyoidean and branchial arteries varies among Chondrichthyes, with most differences observed at the order level. Variation at the family level is less common and occurs in only a few instances. Representatives for three out of the six families of Squaliformes were examined, with Squalus albicaudus differing from other squaliforms in five out of the 16 examined characters (characters 1, 3, 14, 15 and 16). For Squatiniformes, the two species examined showed no variation between characters. Seven out of the ten families of Carcharhiniformes were examined and had variation in regards to the relative position of the second (ch. 16) and third afferent (ch. 11) branchial arteries along the ventral aorta, and the orientation of the fourth artery branching off the aorta (ch. 12). The two Hexanchiformes species examined belong to the family Hexanchidae and did not show any variation. Representatives of four of the eight families of Lamniformes show some variation, with Isurus oxyrinchus (Lamnidae) differing in characters 7 and 8, and Carcharias taurus (Odontaspididae) differing in character 1 and 16, in comparison to the other lamniforms examined. Orectolobiformes, Heterodontiformes, and Pristiophoriformes were represented by a single taxon each, not allowing any evaluation of the morphological variation at the order or family level.

Representatives of the 11 out of the 12 currently recognized families of the Myliobatiformes were examined, and variation was found in the relative position of the second (ch. 16) third (ch. 15) and fourth (ch. 14) afferent branchial arteries, and the orientation of the fourth artery branching off the aorta (ch. 12). In addition, both Gymnura species, Trygonoptera galba, and Plesiobatis daviesi were the only myliobatiforms that had a common trunk of the ventral aorta for the third and fourth afferent branchial arteries. The presence of a dorsal cranial coronary artery was observed only in Rhinoptera bonasus and Mobula hypostoma, with R. bonasus also showing a ventral cranial coronary artery. Mobula hypostoma also has a variation in character 10 and 11, with all arteries equally spaced among each other.

In contrast, the 19 species representing all four families of the Rajiformes did not show any variation except for Sympterygia bonapartii, which is the only rajiform to display the third and fourth afferent arteries splitting from a common branch, separated from the second artery. This branching pattern is similar to what is observed for Rhinopristiformes, whereas Pristis pectinata differs by having the second and third arteries splitting from a common branch. Representatives of three out of the four families in Torpediniformes (all except Hypnidae) were examined and all three Holocephali families were represented.

Soares and Toledo-Piza (2021) described the second and third arteries splitting from a common branch as a synapomorphy for Rajiformes; this character-state was also found in Pristis pectinata (Rhinopristiformes). Amblyraja radiata and Sympterygia bonapartii differ from all other rajiforms by having the third and fourth arteries positioned closer together (6, 0). Additionally, Allis (1912a) described a distinct branching pattern for Raja erinacea (= Leucoraja erinacea [Mitchill, 1825]), with the second, third, and fourth arteries branching all from the same point through a common trunk of the ventral aorta, not exhibiting the pattern previously described for Rajiformes.

Intraspecific variation and bilateral asymmetry were observed in some taxa. For instance, our specimen identified as Amblyraja radiata showed a distinct pattern, with one side showing the three posteriormost arteries branching from the same point of the common trunk, while the other side displays the third and fourth afferent arteries originating from a common branch (6, 0). Another variation in the pattern observed for Amblyraja radiata was illustrated by Satchell (1999) in which the three posteriormost arteries arise from the ventral aorta separately from each other. This asymmetry is also present in both examined specimens of Urobatis jamaicensis and Pateobatis bleekeri, with the second afferent branchial arteries having two distinct ramification points in the ventral aorta, one being lower than the other, which is corroborated by the illustration provided by Kobelkowsky (2012). The distinct patterns of ramification observed by all three authors for Amblyraja radiata could represent either cases of intraspecific variation, inaccurate illustrations provided by previous authors or simply misidentification of the examined specimens.

The presence of a common trunk that branches into the second, third and fourth arteries is shared between Rajiformes, Rhinopristiformes, and Torpediniformes (except Tetronarce puelcha) (4, 1). This character was previously proposed by Corrington (1930) and Daniel (1934) as the “general pattern of batoids”, and it was recovered here as a synapomorphy for Batoidea. However, this character is absent in Myliobatiformes, pointing to a secondary loss and reversion to the ancestral state, in which all three posteriormost afferent branchial arteries branch out separately from the ventral aorta. Additionally, there is difference in the ramification pattern of Tetronarce spp. we observed and that in the illustrations of Tetronarce nobiliana in Satchell (1999), that show the second, third and fourth afferent branchial arteries splitting from a common trunk; this condition disagrees with our observations and those of Soares and Toledo-Piza (2021), where only the third and fourth afferent arteries branch from a common trunk, and the second afferent artery branching independently from the ventral aorta (3, 1). This could indicate variation within the genus. Absence of the common trunk for the three posteriormost arteries is potentially an autapomorphy for T. puelcha, the only torpediniform with this condition.

Another difference from all other examined taxa exhibited by Tetronarce puelcha is the presence of a significantly shorter ventral aorta (ch. 2, state 0), an uncommon character state; that was already observed by Miyake and McEachran (1991) in T. californica and by Satchell (1999) in T. nobiliana. This character state was proposed as an autapomorphy for the genus Tetronarce by Soares and Toledo-Piza (2021), which is corroborated by the present study. Further studies focused on the variation of the ramification pattern in Tetronarce species along their life stages may help to better understand the nature of these unique characters.

Most Myliobatiformes share a branching pattern in which the second, third and fourth branchial afferent arteries branch independently from the ventral aorta. This condition was previously described for Myliobatiformes by Miyake and McEachran (1991), and Kobelkowsky (2012) including the following species: Dasyatis sabina (Lesueur, 1824), Myliobatis sp., Urobatis jamaicensis, Urotrygon munda Gill, 1863, and Urolophus paucimaculatus Dixon, 1969. However, there are a few exceptions to this pattern among myliobatiforms, as in Gymnura spp., Trygonoptera galba, Urolophus viridis and Plesiobatis daviesi, all of which have a common trunk off the ventral aorta for the third and fourth arteries (ch. 3, state 1; Miyake and McEachran 1991).

The branching patterns observed in Myliobatiformes are not exclusive to the group, because they are shared with several of the examined sharks. The condition in which the fourth and third afferent branchial arteries branch from a common trunk off the ventral aorta was observed in the shark species Scyliorhinus haeckelii, Squalus spp., Ginglymostoma cirratum, Heterodontus francisci, Pristiophorus japonicus, and both Squatina species. Among the shark and batoid species examined that had a common trunk from which the third and fourth afferent arteries branch, only Squatina has a considerably longer branch (ch. 5, state 1), which was interpreted by Soares and Toledo-Piza (2021) as an autapomorphy for this genus. The presence of a common trunk for the third and fourth arteries was also observed by Parker and Haswell (1897) in Chiloscyllium modestum (= Brachaelurus waddi [Bloch & Schneider, 1801]), however, this needs further confirmation. Additionally, in previous studies this ramification pattern has also been reported for Centrophorus calceus (= Deania calceus [Lowe, 1839]) (Woodland 1906). Parker (1886) described this branching pattern for Mustelus antarcticus Günther, 1870, however the specimen of Mustelus canis examined herein do not have the common trunk that branches into the third and fourth arteries, but both arteries branch very close to each other, a condition also illustrated by Ferguson (1911: 177, fig. 5).

In contrast, the most common configuration found in Selachii, similar to Myliobatiformes, is the second, third and fourth afferent arteries branching independently off the ventral aorta, a condition also observed by O'Donoghue and Abbott (1928). The same condition was found in the carcharhiniforms Galeus glaucus (= Carcharhinus glaucus) and Cestracion zygaena (= Sphyrna zygaena), the lamniform Carcharias littoralis (= Odontaspis taurus [Rafinesque, 1810]), and the hexanchiforms Heptanchus cinereus (= Heptranchias perlo), Notorynchus and Chlamydoselachus (Allis 1912b, 1912c; Corrington 1930; Daniel 1934).

Allis (1912b) showed Heptanchus cinereus (= Heptranchias perlo) as having the first common trunk absent, with the hyoidean and first afferent branchial arteries branching independently off the ventral aorta, which contrasts with our observations. Soares and Toledo-Piza (2021) claimed that Ferguson (1911) also reported/illustrated the same pattern, with both the hyoidean and first afferent branchial arteries branching independently for H. maculatus (= Notorynchus cepedianus). After studying the illustration provided in the original article, however, it was confirmed that the branching pattern is similar to the one described here for Heptranchias, i.e., the presence of a first common trunk. The absence of a first common trunk is exclusive to Holocephali, with all three examined specimens having the hyoidean and first afferent branchial arteries branching separately off the ventral aorta. This is corroborated by Allis (1912c), where Chimaera colliei (= Hydrolagus colliei) is illustrated with both arteries arising independently. Therefore, it is possible that the absence of a first common trunk is a synapomorphy for Holocephali.

The maximum parsimony trees resulting from the addition of our afferent branchial characters to the original matrices of Aschliman et al. (2012a) and Shirai (1996) showed differences when compared to the original topologies published by these authors. A key difference between the hypothesis recovered here and Aschliman et al. (2012a) regards the phylogenetic placement of Rhinopristiformes species included in the analysis. While our hypothesis recovered Pristis pectinata and Zapteryx exasperata deriving together and Trygonorrhina fasciata and Rhinobatos productus as a polytomy with Rajiformes, the tree in Aschliman et al. (2012a) recovered well resolved branches with Pristis pectinata as the first to derive. Despite these differences, both studies recovered Rajiformes as a derived branch within this group, which may be supported by the ramification pattern shared by Rhinopristiformes and Rajiformes in which the three posteriormost afferent branchial arteries branch from the ventral aorta through a common trunk. The monophyly of Myliobatiformes as well as their position as the most derived clade of Batoidea is supported by several morphological and molecular phylogenetic studies (McEachran and Aschliman 2004; Naylor et al. 2016; Naylor 2025).

The most notable differences between the hypothesis recovered here and the one presented by Shirai (1996) is a polytomy including all representatives of Etmopteridae, and the sister-relationship of gymnurids and the clade formed by dasyatids/urolophids and all other myliobatiforms. The phylogenetic placement of gymnurids was supported by one character in our analysis: the long distance between the anterior tip of the ventral aorta and the anterior end of the conus arteriosus (ch. 2, state 0), being recovered as a synapomorphy for the group. The absence of a common trunk of the ventral aorta for the third and fourth afferent branchial arteries (ch. 3, state 0) was recovered as a synapomorphy for Batoidea in both maximum-parsimony analyses. Since this character is exhibited in only a few of the examined species in Myliobatiformes, this condition could have been secondarily lost in several branches.

This study increases the information available about the morphological variation in the afferent branchial arteries within Chondrichthyes, through the examination of a considerable number of chondrichthyan taxa. Due to the application of a broader comparative framework, we were able to refine the understanding of these anatomical structures and their potential evolutionary implications. The branching pattern for the afferent branchial arteries was described for each order and character states were mapped to assess the evolutionary history of these structures. In most taxa examined, the three posteriormost arteries branch independently from the ventral aorta with the exception of representatives of Rajiformes, Rhinopristiformes, and Torpediniformes; this latter pattern may be related to the dorsoventral flattening of the body in batoids and could indicate differences in blood supply to the gills but further investigation is needed. Through a larger diversity of shark taxa examined, we were able to identify a new structure described as a ventral coronary cranial artery. Nevertheless, it is noteworthy that an even larger taxonomic sampling would be desirable to achieve a more comprehensive understanding of the evolution of afferent branchial arteries and other heart structures and their implications in the chondrichthyan interrelationships. Thus, we emphasize the need for more comprehensive anatomical studies and integrative phylogenetic and ecomorphological approaches to reconcile existing conflicts.

The authors thank Cristiano Moreira and Marcelo Britto (MNRJ), Hugo Santos (UERJ), Alessio Datovo (MZUSP) and Andrew Williston (MCZ) for permission to dissect specimens under their care. To the artisanal fishermen of Rio de Janeiro for donating specimens for this research. Also to Alastair Graham (CSIRO), Dave Catania (CAS) and Diane Pitassy (USNM) for the loan/donation of specimens. MCEBA was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and KDAS was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, SEI-260003/006392/2024-APQ1 and E-26/204.488/2024). We are grateful for the critical comments of the reviewers and the subject editor, which improved previous drafts of this manuscript.