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Skeletal development in the heterocercal caudal fin of spotted gar (Lepisosteus oculatus) and other Lepisosteiformes

. Author manuscript; available in PMC: 2019 May 1.

Published in final edited form as: Dev Dyn. 2018 Jan 31;247(5):724–740. doi: 10.1002/dvdy.24617

Abstract

Background

The caudal fin of actinopterygians experienced substantial morphological changes during evolution. In basal actinopterygians, the caudal fin skeleton supports an asymmetrical heterocercal caudal fin, while most teleosts have a symmetrical homocercal caudal fin. The transition from the ancestral heterocercal form to the derived homocercal caudal fin remains poorly understood. Few developmental studies provide an understanding of derived and ancestral characters among basal actinopterygians. To fill this gap, we examined the development of the caudal fin of spotted gar Lepisosteus oculatus, one of only eight living species of Holostei, the sister group to the teleosts.

Results

Our observations of animals from fertilization to more than a year old provide the most detailed description of the developmentof caudal fin skeletal elements in any Holostean species. We observed two different types of distal caudal radials replacing two transient plates of connective tissue, identifying two hypaxial ensembles separated by a space between hypurals 2 and 3. These features have not been described in any gar species, but can be observed in other gar species, and thus represent anatomical structures common to lepisosteiformes.

Conclusion

The present work highlights the power and importance of ontogenic studies, and provides bases for future evolutionary and morphological investigations on actinopterygians fins.

Keywords: Holostei, actinopterygian, tail, ontogeny, skeletogenesis, morphology

Introduction

The Actinopterygii (ray-finned fishes) is the most species-rich group of vertebrates, including over 32,000 living species, including about half of all extant vertebrate species (Eschmeyer, 2015; Faircloth et al., 2013; Nelson, 2006; Sallan, 2014). Within ray-finned fishes, an important evolutionary step was an improvement of swimming capabilities by the acquisition of more delicate control of dorsal and anal fin movements that appeared with the neopterygian fish lineage (literally meaning “fish with new fins”), a group that includes gars, bowfin, and teleost fishes. Evolution of the dorsal and anal fins occurred concomitantly with other morphological innovations, in the skull and mouth for example, which provided improvements in feeding capacities leading to the rise of the neopterygians as the taxonomically dominant group of vertebrates (López-Arbarello, 2012). Among neopterygians, the largely dominant Teleost infraclass represents more than 99.9% of living actinopterygian species (Eschmeyer, 2015; Nelson, 2006). Teleosts are characterized morphologically by numerous lineage-specific characters, including additional innovations in the jaw that further improved feeding, as well as the emergence of a homocercal caudal fin (Arratia, 2015; Sallan, 2014; Schultze and Arratia, 1989; Schultze and Arratia, 2013), which may have contributed to the successful radiation of teleosts into almost all aquatic ecosystems. Indeed, the caudal fin is a major component of mobility in fish because it generates swimming power and contributes to maneuverability, thus helping to control swimming speed and direction of movement, which facilitate prey capture and predator avoidance (Flammang and Lauder, 2009; Lauder, 2000). The caudal fin and its supporting internal skeleton are highly variable among actinopterygians, so different skeletal elements, their morphological variations, and their associations with each other are frequently used in a variety of ecological, biomechanical, and systematic studies (eg. (Arratia and Schultze, 1992; Cloutier et al., 2011; Cloutier and Arratia, 2004; Fiaz et al., 2012; Flammang and Lauder, 2009; Grünbaum et al., Lauder, 2000; Schultze and Arratia, 1986; Schultze and Arratia, 1988; Schultze and Arratia, 2013)).

Exteriorly, the homocercal caudal fin of extant teleosts generally appears to have a dorso-ventral symmetry and differs from the caudal fin of most non-teleost ray-finned fishes, which have a heterocercal caudal fin that is generally asymmetrical, representing the ancestral condition (Metscher and Ahlberg, 2001; Moriyama and Takeda, 2013; Schultze and Arratia, 1989). The notion of homocercality, however, is not directly related to symmetry, but instead is defined by the terminus of the notochord: in the teleost homocercal caudal fins, the notochord ends shortly after the posterior margin of the most posterior endoskeletal element and does not extend along the dorsal margin of the first principal caudal-fin ray, but in heterocercal caudal fins, the notochord extends beyond the most posterior endoskeletal element along the dorsal margin of the first principal caudal-fin ray (Arratia, 2015). Although gars and bowfin have a superficially symmetrical caudal fin formed by one caudal fin lobe, these fishes nevertheless have a heterocercal caudal fin because the notochord extends to the tip of the fin on the dorsal side of the most dorsal principal ray.

Despite numerous studies on caudal fin skeletons (e.g. (Metscher and Ahlberg, 2001; Moriyama and Takeda, 2013; Schultze and Arratia, 2013)), the morphological and genetic processes by which the caudal fin transitioned from the heterocercal state in basal actinopterygians to the homocercal form in teleosts remains not well understood. This information gap is due largely to several factors. 1) The study of the actinopterygian skeleton through evolutionary time relies mostly on fossils, and thus almost entirely on mineralized adult specimens. 2) In fossils, caudal fin endoskeletal elements are not always complete or visible, for example, they are often covered by fossilized scales in Neopterygians. 3) Despite the richness of the fossil record, evolutionarily intermediates or novel lineages are rare and difficult to classify with respect to other described lineages. 4) Finally, surviving lineages of non-teleost actinopterygians are depauperate: from the ray-finned fish diversity that appeared sometime between the emergence of actinopterygians about 400 million years ago (mya) and the emergence of teleosts about 330–270 mya (Arratia, 2015; Near et al., 2012; Sallan, 2014), only 49 species remain (12 species of polypteriformes, 29 species of acipenseriformes, and 8 species of holostei) limiting considerably the number of species available for controlled developmental studies. The rarity of younger, cartilaginous, or partially mineralized developmental stages in fossils precludes detailed studies of skeleton ontogeny, which in return could inform about common developmental processes and transitory characters that are no longer present or visible in adults, but are retained in embryos or larvae as vestiges of developmental processes shared by common ancestors (Hall, 2003; Wanninger, 2015). Detailed comparative analyses of a variety of actinopterygians are thus necessary to better understand the developmental and evolutionary mechanisms involved in the changes of the ray-finned fish caudal fin.

The few available studies on the gar caudal fin skeleton are mostly based on adult specimens of either of the two gar genera, Lepisosteus or Atractosteus. The first detailed description of the gar caudal fin appeared in the seminal work of Nybelin (1977), who studied the skeleton of two adult specimens of longnose gar (Lepisosteus osseus) along with bowfin and some other actinopterygians species. The caudal skeletons of gar and bowfin were reevaluated by Schultze and Arratia (1986), who highlighted the similarities and differences of these species based on a dozen juvenile and adult individuals of longnose and shortnose (Lepisosteus platostomus) gars. Soon after, Bartsch briefly described the gar caudal skeleton based on one juvenile Florida gar (Lepisosteus platyrhincus) and one adult longnose gar in comparison to other actinopterygian skeletons (Bartsch, 1988). Finally, Grande (2010) published a detailed analysis of the gar skeleton, including a description of the caudal fin skeleton in young and adult longnose gars based on 45 specimens, and alligator gars (Atractosteus spatula) based on 44 specimens. These studies, however, did not pursue a strong developmental perspective focusing on the formation, growth, and developmental relationships of caudal fin skeletal elements.

Using spotted gar as a representative holostean (Betancur-R et al., 2013; Betancur-R et al., 2017; Braasch et al., 2015; Eschmeyer, 2015; Faircloth et al., 2013; Nelson, 2006; Wright et al., 2012), we analyzed the development of the caudal fin skeleton and compared it to the caudal fin of other gar species to set the stage for future evolutionary, morphological and functional studies enabled by the release of the reference genome sequence and the emergence of developmental genetic tools (Braasch et al., 2015; Braasch et al., 2016). We raised spotted gar embryos and larvae from fertilization to more than a year old and sampled them regularly during development, supplemented with a few wild-caught adult spotted gar from Louisiana. Our ontogenetic investigations of the spotted gar caudal fin skeleton provides 1) a thorough description of the developmental pattern and timing of caudal fin skeletal elements; 2) a comparison of the spotted gar caudal fin skeleton to those of other gar species; and 3) the description of previously undescribed and conserved structures in the gar caudal fin skeleton. These studies reveal the evolutionary conservation of two different types of distal caudal radials that replace two transient plates of connective tissue, identifing two hypaxial ensembles separated by a space between hypurals 2 and 3 as a common feature of gar and teleost caudal fins. These findings suggest ancient developmental modules that likely contributed to the evolution of the neopterygian and teleost caudal fin.

Results and Discussions

Overview of spotted gar caudal fin development

Externally, the posterior portion of the embryonic fin-fold in newly hatched spotted gar embryos and early larvae (5–6 dpf and ~8 mm total length (TL), Fig. 1D) appeared roughly symmetrical dorso-ventrally from the slightly up-turned notochord (abbreviated nc in Fig. 3A). By stage 31 (8–9 dpf and ~10 mm TL, Fig. 1D) (Braasch et al., 2015; Grande, 2010; Long and Ballard, 2001), asymmetry and patterning of the different median fins became visible within the fin-fold (ff, marked by dotted lines in Fig. 3B) due to the pigmentation pattern, which demarcated the future anal, dorsal, and caudal fins (Fig. 3B), and due to the presence of actinotrichia both in the epichordal and hypochordal lobes of the caudal fin. The fin-fold subsequently subdivided into the dorsal, anal, and caudal fin-folds and the caudal fin-fold became highly asymmetrical as the notochord extended caudally past the hypaxial elements and flexed slightly upward when the larvae reached about 22–24 mm SL (~25 dpf and ~30–32 mm TL, Fig. 1D), thereby forming the opisthural lobe (Fig. 3C, ol), a morphological feature first described in 1856 (Agassiz, 1877) and previously referred as the notochordal appendage (Carpenter, 1975); all extant gar species conserve this feature (Grande, 2010). Shortly later, at about 25–30 mm SL (~27 dpf and 32–35 mm TL, Fig. 1D), the epichordal caudal fin-fold started to regress (Fig. 3C) and dorsal fulcra later developed in that location (Fig. 3D, fub). The opisthural lobe remained as a protruding fin-like structure emerging from the dorsal base of the caudal fin lobe and lying superior to it until animals were almost a year post fertilization. Because the notochord extension can make rapid oscillations, it has been hypothesized to provide enhanced mobility, improving predatory behavior of young gars (Carpenter, 1975). At around 150–200 mm SL, the opisthural lobe started to disapear (Fig. 3D), became shorter than the most dorsal caudal fin ray, and was usually unidentifiable at around 250 mm SL (Fig. 3E), similar to what has been reported for other gar species (Carpenter, 1975; Grande, 2010). In adults, after the disappearance of the opisthural lobe and the development of caudal fin rays, at about a year or around 250 mm SL, the single lobe of the caudal fin was convexly rounded and the margin formed an almost symmetrical shape (Fig. 3E, dotted line). Asymmetry of the caudal fin was still evident, however, due to the oblique orientation of the posterior border of the caudal peduncle, which was more anterior ventrally and more posterior dorsally at the position of the resorbed opisthural lobe (Fig. 3E, dashed line).

Figure 1.

Figure 1

Spotted gar caudal fin organization and development. (A) Caudal portion of a skeletonized adult spotted gar (471 mm SL), left view. (B) Schematic representation of the spotted gar caudal fin skeleton, left view. (C) Sample distribution by 5 mm size classes (Standard Length (SL) in mm) and by type of staining: green, Alcian Blue + Alizarin Red; blue, Alcian Blue only; red, Alizarin Red only. (D) Age-length relationship of developing spotted gars from 7 days post fertilization (dpf) and 121 dpf. The black solid line represents the average total length (TL, in mm) and the dashed blue lines represent ± one standard deviation (SD) from the average TL. Abbreviations: c, centrum; cfr, caudal fin rays; darc, dorsal arcualia; dcr, distal radials; ep, epural; fub, basal fulcra; fufr, fringing fulcra; ha, haemal arch; hs, haemal spine; hyp, hypural; na, neural arch; nsp, paired neural spines; phy, parhypural; pp, parapophysis; pu, preural centra; u, ural centra.

Figure 3.

Figure 3

External view of spotted gar caudal fin development. (A) 7.5 mm TL, equivalent Stage 25 (Braasch et al., 2015; Long and Ballard, 2001). (B) 13 mm TL, equivalent Stage 31. (C) 22.5 SL, 30.5 mm TL. (D) 147 mm SL, 174 mm TL. (E) 200 mm SL, 252 mm TL. The dotted line in (B) denotes the margin of the larval fin fold. The dotted line in (E) highlights the almost symmetrical shape of the adult spotted gar caudal fin. The dashed line demarcates the oblique delimitation of the boundary between the scaled caudal peduncle and the caudal fin rays, which contribute to most of perceived asymmetry. Abbreviations: af, anal fin; av, anal vent; df, dorsal fin; el, epichordal lobe of the caudal fin; ff, fin fold; fub, basal fulcra; hl, hypochordal lobe of the caudal fin; int, intestine; ol, opisthural lobe; nc, notochord; y, yolk.

Internally, between the posterior end of the anal fin and the anterior extent of the caudal fin, the caudal skeleton of the developing spotted gar was generally organized around four to six caudal centra supporting haemal arches (Fig. 1A insert, abbreviations c and ha, respectively). The caudal fin skeleton was set up around six or seven preural centra (pu) and up to seven mineralized ural centra (u), each supporting ventrally a haemal spine (hs) or a hypural element (hyp) respectively (Fig. 1A–B). Neural arches (na) and neural spines (nsp) formed dorsally on all centra, but were greatly reduced in the ural part of the caudal fin (Fig. 1A–B). Fully developed specimens usually had five epurals positioned in the mesenchyme above the ural centra (Fig. 1A–B). The notochord exhibited a slight flexion into the upper lobe around ural centrum 1 (Fig. 1A–B).

The first sign of cartilage formation in caudal skeleton development, as determined by the median value of Alcian uptake, occurred by 12 mm SL in the first and second hypural rudiments, followed shortly by Alcian uptake in the parhypural (phy) and haemal elements (Fig. 2, Fig. 4A–C) and in basiventrals (bv) around 15 mm SL (~20 mm TL and 17–18 dpf, Fig. 1D, Fig. 2, Fig. 4C). The upturning of the notochord into the upper lobe, although slight, occurred around 20–25 mm SL (~28–33 mm TL and 25–28 dpf, Fig. 1D), near hypural 1 at the expected position of ural centrum 1, and was concomitant with the first few hypurals achieving their final cartilaginous shape. The epurals became Alcian positive around 21 mm SL (~28–30 mm TL and 25 dpf, Fig. 1D, Fig. 2, Fig. 4D), followed later by mineralization of the dorsal and ventral basal fulcra (fub) around 38 and 101 mm SL respectively (~43 mm TL and 34 dpf, and ~125 mm TL and 120 dpf, respectively, Fig. 1D, Fig. 2, Fig. 4E). The last caudal fin skeletal elements to form were the centra, with the first ural centrum mineralizing around 50 mm SL (~60 mm TL and 50 dpf, Fig. 1D, Fig. 2, Fig. 4E). Except for ural centrum 2 and centra further posterior, by 155 mm SL, all structures were as developed and ossified as in the largest adult individual (416 mm SL). These most caudal ural centra continued to ossify slowly after 155 mm SL, but even in the largest individual we studied, the final few ural centra had not mineralized completely and some dorsal arcualia (darc) continued to show only Alcian staining (Fig. 1A–B, Fig. 2, Fig. 4E–F). The sequence of development using the average and the fastest growing individuals are shown in Supplemental Figure 1 and 2 respectively.

Figure 2.

Figure 2

Sequence of development and ossification of individual caudal fin skeletal elements based on median values. Diamonds indicate earliest appearance of elements by cell condensation. Blue indicates Alcian Blue uptake and development of cartilage. The blue-to-red transition represents the progression of ossification. Red represents fully mineralized structures. Less opaque bars refer to elements found in less than 25% of the studied specimens.

Figure 4.

Figure 4

Developmental sequence of the spotted gar caudal fin skeleton. (A) 12 mm SL. (B) 13 mm SL. (C) 15 mm SL. (D) 21 mm SL. (E) 62 mm SL. (F) 416 mm SL. The white elongated triangle indicates hypural 1 as a reference point. Scale bars indicate 1 mm. Abbreviations: bv, basiventral arcualia; cfr, caudal fin rays; darc, dorsal arcualia; ep, epural; fub, basal fulcra; fufr, fringing fulcra; hs, haemal spine; hyp, hypural; nc, notochord; pu1, preural centrum 1; u, ural centra; u1, ural centrum 1.

Detailed development of gar caudal fin skeletal elements

Hypurals

In actinopterygians, hypurals are flanked laterally by caudal blood vessels and possess a single proximal attachment to the notochord (Schultze and Arratia, 1988; Schultze and Arratia, 1989; Bensimon-Brito et al., 2010; Bensimon-Brito et al., 2012; Grande, 2010). In our samples, hypurals developed as endochondral bones, forming into cartilage first and later ossifying perichondrially into bone (Fig. 4, Fig. 5A–C). The first hypural (hyp1) formed as a sturdy cartilaginous anlage on or close to the notochord at around 9 mm SL (Fig. 2, Fig. 4A) and more posterior hypurals developed serially in a similar pattern with the posterior most hypurals, hypurals 5 and more posterior, generally starting to take up Alcian at around 23 mm SL (Fig. 2, Fig. 4A–C, Fig. 5B–C, M). Hypurals developed proximo-distally. Usually seven hypurals developed in our spotted gar specimens, with a range from four to 11 (Fig. 6A–B). Grande (2010) described the presence of six to eight hypurals, most frequently eight, in spotted gar (Table 3). The greater variation observed among our samples is likely due to the much larger number of individuals studied here and/or differences in captive rearing conditions potentially affecting the skeletal development, as observed in some other situations (e.g. (Boglione et al., 2013a; Boglione et al., 2013b)). The number of hypurals in spotted gar also appears to be similar to the number observed in other gar species, e.g., ten in longnose gar (Nybelin, 1977), seven to twelve in longnose and shortnose gars (Schultze and Arratia, 1986), and six to eleven if all seven extant gar species are considered (Grande, 2010) (Table 3). In our animals, hypurals continuously decreased in size posteriorly and the posterior-most hypurals remained as non-mineralized cartilages in many adults and were generally small, less elongated, or rounded and disconnected from the notochord (Fig. 4E, Fig. 6B), as observed in other gar species (Bartsch, 1988; Grande, 2010; Schultze and Arratia, 1986). For both longnose gar and shortnose gar, the disconnection of posterior hypurals from the notochord has been suggested to be caused by the upward flexion of the notochord and the dorsal-to-ventral mineralization of the ural centra resulting in a failure of centra to fuse to hypurals (Schultze and Arratia, 1986). The same mechanisms could also explain the separation of terminal hypurals and the notochord observed in spotted gar. Hypural 1, and sometimes hypural 2, had smaller articulating processes on their proximal epiphyses along the notochord compared to more posterior hypurals, which extended antero-ventrally on the ventral surface of the centra (Fig. 5B–C), in agreement with observations by Nybelin (1977).

Figure 5.

Figure 5

Detailed development of major caudal fin skeletal elements in spotted gar. (A) 16 mm SL, sagittal section. (B) 13 mm SL. (C) 61 mm SL. (D) 12 mm SL. (E) 10 mm SL. (F) 14 mm SL. (G) 60 mm SL. (H) 61 mm SL. (I) 79 mm SL. (J) 17 mm SL. (K) 23 mm SL. (L) 37 mm SL. (M) Schematic representation of developmental dynamics of caudal fin skeletal elements. The origin of the arrow marks the first element to form and the arrowhead indicates the direction of developmental progression. The white elongated triangle indicates hypural 1 as a reference point, and the black arrow points at the gap between the two plates of connective tissue and the change of size and shape in hypurals. Small equilateral white triangles indicate haemal elements supporting caudal fin rays. Small black equilateral triangles indicate haemal elements not supporting caudal fin rays. Scale bar in (A) indicates 100 μm; other scale bars indicate 1 mm. Abbreviations: bv, basiventral arcualia; darc, dorsal arcualia; dcr, distal caudal radials; ep, epural; fub, basal fulcra; ha, haemal arch; hs, haemal spine; hyp, hypural; hyp1, hypural 1; na, neural arch; nc, notochord; ns, neural spine; phy, parhypural; pp, parapophysis; pu1, preural centrum 1; u1, ural centrum 1.

Figure 6.

Figure 6

Variation in hypurals and epurals among gar individuals. (A) 165 mm SL. (B) 58 mm SL. (C) 219 mm SL. (D) 75 mm SL. (E) Regression of the number of epurals against the number of hypurals in individuals having all skeletal elements formed. The size of each circle denotes the number of specimens displaying indicated number of hypurals and epurals. (F) 103 mm SL. In (A), (B) and (F), the white elongated triangle indicates hypural 1; in (A) and (B), the grey elongated triangle points at hypural 2 and more posterior hypurals. Scale bars indicate 1 mm. Abbreviations: avp-hyp1, antero-ventral process of hypural 1; ep, epurals; hyp, hypurals.

Table 3.

Counts of caudal fin skeletal element in gar species

Species Hypurals Haemal Spines Epurals Lepidotrichia Ural centra Reference
L. oculatus (Spotted gar) 4 to 11 6 or 7 4 to 7 12 4 to 7 Present study
4 to 6 N/A 4 or 5 12 7 to 9 Grande. 2010
L. osseus (Longnose gar) 8 to 11 7 or 8 * 4 to 8 12 6 to 8 Grande. 2010
10 8 5 12 7 or 8 Nybelin, 1977
7 to 12 10 5 to 8 12 N/A Schultze & Arratia, 1986
L. platostomus (Shortnose gar) 8 to 10 N/A 5 to 7 11 to 13 6 to 9 Grande. 2010
7 to 12 10 5 to 8 11 or 12 N/A Schultze & Arratia, 1986
L. platyrhincus (Florida gar) 6 to 9 N/A 4 or 5 11 or 12 6 to 9 Grande. 2010
A. spatula (Alligator gar) 7 to 10 7 or 8 * 4 to 6 12 8 to 12 Grande. 2010
A. tristoechus (Cuban gar) 7 to 9 N/A 4 or 5 12 6 to 7 Grande. 2010
A. tropicus (Tropical gar) 7 to 9 N/A 3 to 6 12 7 to 9 Grande. 2010

Mineralization of hypural elements began after these cartilaginous structures had acquired their general shape. The first signs of mineralization in hypurals occurred in the diaphysis, or shaft, of hypural 1 around 22 mm SL (~30 mm TL and 25 dpf, Fig. 1D, Fig. 2). Ossification then progressed both toward and away from the notochord (Fig. 4D). The epiphyses, or extremities, of the hypurals were the last part of the hypurals to mineralize, with caps of non-mineralized cartilage remaining on the proximal end (Fig. 4E). The sequence of mineralization mirrored that of cartilage formation, beginning with hypural 1 and progressing posteriorly with mineralization continuing on each following hypural before the hypural anterior to it had fully ossified. The final size of each respective hypural decreased posteriorly, and mineralization of most hypurals was completed by about 90 mm SL (~110 mm TL and 110 dpf, Fig. 1D, Fig. 2). In a number of large specimens, including the largest individual studied (416 mm SL), however, the most posterior hypurals remained cartilaginous (Fig. 2). In one of our fully developed individuals, hypurals 1 and 2 were partially fused (data not shown) and in 12 fully developed specimens, hypural 1 harbored a small process projecting antero-ventrally near the proximal epiphysis and articulating with the parhypural (Fig. 6F), similar to a previously described longnose gar specimen (Nybelin, 1977) and drawn, but not mentioned, in two specimens of shortnose gar (Fig. 7 in (Schultze and Arratia, 1986)), and similar to the anterior process often observed in some teleost species such as salmonids (Arratia and Schultze, 1992; Grünbaum and Cloutier, 2010).

In our developing and adult specimens, hypurals were generally evenly spaced and were progressively smaller posteriorly, except for hypurals 2 and 3, which differed more in size and shape than other hypural neighbor pairs and generally had a larger space between them (Fig. 5B–C, J–L). The magnitude of the separation between hypural 2 and 3 and size and shape differences varied among specimens, and while we could find no previous report or discussion of this feature by other authors, our revisiting of published figures, drawings, and photographs revealed similar features of caudal skeletons in different gar species (e.g. in longnose gar in Fig. 1A in Nybelin (1977), Fig. 2A and Fig. 6 in Schultze and Arratia (1986), Fig. 91 in Grande (2010), and in Florida gar in Fig. 24A in Bartsch (1988), although note that the latter figure mis-labels hypural 1 due to the fusion of vertebral centra pu2 and pu3 in that particular specimen of Florida gar, and therefore all other hypaxial elements are also misnumbered). We conclude that the spacing between hypurals 2 and 3, and the greater change in size and shape of neighboring hypurals for this pair compared to other hypural neighbor pairs represents a general organization pattern common among gar species but is described here for the first time.

Haemal spines

The haemal spines and haemal arches, like hypurals, formed as endochondral bones ventral to the preural centra (Fig. 4, Fig. 5D–F). We numbered haemal spines following the usual nomenclature by starting with haemal spine 1 being the parhypural, located just anteriorly of hypural 1, and increasing in numbered designation anteriorly because, unlike hypurals, haemal spines develop serially from posterior to anterior (Fig. 2, Fig. 4A–C, Fig. 5D–F, M). Almost simultaneously, haemal spines 1 to 4 formed as cartilaginous anlagen in the mesenchyme slightly ventral to, but not touching, the notochord at around 11 mm SL (Fig. 2, Fig. 5A, Fig. 5D). Haemal elements 1 to 6 or 7 formed initially as spines that further developed arches proximal to the preural centra, creating the haemal arch in articulation with the basiventral arcualia (Fig. 5D–F, white triangles). The same six or seven haemal elements also supported caudal fin rays and thus the most anterior haemal element delimitated the anterior margin of the caudal fin skeletal ensemble (Fig. 1B, Fig. 5F). In contrast, we show for the first time that the later developing haemal elements (haemal element 7 or 8 and any more anterior) developed initially into haemal arches that later extended distally into spines (Fig. 5F, black triangles). Mineralization of haemal elements occurred in a fashion similar to the hypurals, beginning as a ring on the diaphysis and progressing toward the epiphyses, with the first signs of mineralization seen almost simultaneously on haemal element 3 to 7 around 22 mm SL (~28–30 mm TL and 18–19 dpf, Fig. 1D, Fig. 2). The most anterior haemal spine studied completed ossification around 70 mm SL (~90 mm TL and 83–85 dpf, Fig. 1D, Fig. 2).

All fully developed individuals examined had eleven or twelve haemal elements between those articulating with the anal fin proximal elements anteriorly and the hypurals posteriorly (Fig. 2). Usually six or seven haemal spines supported the caudal fin rays and are thus part of the caudal fin skeleton, which is in agreement with the reported number of eight caudal fin haemal spines in longnose gar (Nybelin, 1977), up to ten in the longnose and shortnose gars (Schultze and Arratia, 1986), eight drawn for Florida gar (Bartsch, 1988), and seven or eight in both the longnose and alligator gars (Grande, 2010) (Table 3). The four to six studied anterior haemal elements, which do not support caudal fin rays and thus are part of the caudal skeleton and not of the caudal fin skeleton (Fig. 1B), were distinctly shorter and thinner than the caudal fin haemal elements (Fig. 1A–B, Fig. 4D–F, Fig. 5F). About 7% of the specimens among our samples showed a partial fusion of one pair of adjacent haemal spines, occurring between the parhypural and haemal spine 2 in four individuals, between haemal spines 2 and 3 in six individuals, or between haemal spines 3 and 4 in one individual (data not shown). Partial fusion of the parhypural and haemal element 2 has also been seen in a longnose gar specimen (Schultze and Arratia, 1986). The variety of partial fusions observed in the present study, compared to examples previously reported, is likely due to the much larger number of specimens studied here and perhaps to the rearing conditions.

Basiventral arcualia and parapophyses

The paired basiventral arcualia, anlagen of the vertebrae parapophyses that articulate with the haemal arches, developed ventrally and in direct contact with the notochord and were numbered in the same manner as the associated haemal elements (Fig. 2, Fig. 4C, Fig. 5A). Basiventrals 6 through 8 generally formed first, at around 11 mm SL (~14–16 mm TL and 13–15 dpf, Fig. 1D), beginning as cartilaginous rudiments and increasing in size as they grew connected to the notochord and dorsal to their relative haemal element (Fig. 2, Fig. 5F, 5M). Interestingly, development of the subsequent basiventrals progressed in both anterior and posterior directions, and they later articulated with their respective haemal arches on the ventro-lateral sides of the centra between 18 and 23 mm SL for all basiventrals (Fig. 5F, 5M). Mineralization of basiventrals into parapophyses began at the dorsal end of all basiventral arcualia around 60 mm SL (~78–82 mm TL and 65–70 dpf) and progressed ventrally down the structure (Fig. 1A, Fig. 2, Fig. 5G). Fusion of the dorsal portion of each parapophysis with its respective centrum, occurring around 80 to 90 mm SL, took place before the whole structure had completely ossified (Fig. 5H). The number of parapophysis pairs was consistent with the number of haemal elements, either eleven or twelve in different individuals. The last pair of parapophyses to mineralize was fully ossified at around 100 mm SL (~125 mm TL and 120 dpf, Fig. 1D, Fig. 2). Once fully developed, parapophyses articulated with their respective haemal arch, thereby completing the haemal canal. This report represents the first description, to our knowledge, of basiventral and parapophyses formation in gars, and the delineation of the different developmental sequences leading to basiventral and haemal elements.

Centra and vertebrae

Because the development of centra in several gar species has already been well described (Grande, 2010; Schultze and Arratia, 1986), we here aimed to document the general pattern and developmental timing in spotted gar samples without a detailed description of the process of centra mineralization.

In agreement with previously published observations for other gar species (Grande, 2010; Schultze and Arratia, 1986), spotted gar centra mineralized as endoskeletal elements from arcualia, and preural and ural centra showed different patterns of ossification. Dorsal and ventral to the notochord, preural centra began to mineralize from the basidorsal and basiventral arcualia respectively, and mineralization progressed laterally around the notochord to meet roughly at its midline (Fig. 5G). In contrast, mineralization of ural centra began only from the basidorsal arcualia, at around 50 mm SL for ural centrum 1 (~60 mm TL and 50 dpf, Fig. 1D), and progressed laterally around the notochord toward the ventral side of the centrum (Fig. 2, Fig. 5H–I). Neither interdorsal nor interventral arcualia were observed (Fig. 5G–I), which is in agreement with previous observations in other gar species (Grande, 2010; Schultze and Arratia, 1986). Notably, mineralization of preural and ural centra followed a strict antero-posterior progression and the initiation of mineralization of each subsequent centrum occurred before the centra anterior to it had completely ossified (Fig. 2, Fig. 5G–I, M), similar to the pattern reported for longnose and alligator gars (Grande, 2010).

Each preural centrum in spotted gar, except preural centrum 1, associated with a single haemal and neural element (monospondylous condition) (Grande, 2010; Schultze and Arratia, 1986). In contrast, in 71% of our specimens with mineralized centra, preural centrum 1 articulated with both the parhypural and the first hypural (see examples in Fig. 4F and Fig. 5I). Frequent double articulation coupled with the common presence of an extra neural arch on preural centrum 1 and signs of a fusion line in this centrum, led to the postulation of a fusion of the first preural and ural centra in gars (Bartsch, 1988; Grande, 2010; Nybelin, 1977; Schultze and Arratia, 1986). Our analysis does not provide sufficient evidence to evaluate this fusion hypothesis; more directed developmental studies of this centrum are required.

The preural and ural vertebrae in gars were holospondylous meaning that the neural arches eventually fused with their respective ossified centrum to form the vertebrae. In general, each preural vertebra included a pair of parapophyses fused to the preural centrum, along with a neural arch and paired neural spines fused to the dorsal parapophyses and dorsal arcualia (Fig. 5G–I). In agreement with previous observations (Grande, 2010), the most caudal preural vertebra, and sometimes a few anterior preural vertebrae, frequently harbored a unique median neural spine (data not shown). In contrast, the ural centra lacked parapophyses and exhibited an incomplete neural arch and small or absent neural spines (Fig. 1B, Fig. 5I).

The number of ural centra varied from four to seven among our fully developed samples, but because the posterior-most centra were only partially ossified and numerous Alcian-positive basidorsal arcualia followed posteriorly (Fig. 4E, F; Fig. 5I), precise determination of ural centra number and their developmental timeline was difficult.

Neural spines and epurals

Our observations showed that epurals are endochondral bones, in agreement with previous observations (Grande, 2010). While neural arches and neural spines appeared to grow out from the dorsal arcualia; epurals instead condensed in the dorsal mesenchyme from cartilaginous rudiments that developed proximally towards the notochord between the pair of neural spines present on each vertebra (Fig. 4D, Fig. 5J–K). Epurals began forming at around 13 to 19 mm SL as cartilaginous elements in the mesenchyme dorsal to the ural portion of the notochord prior to its flexion, which occurs at around 20 to 25 mm SL. Epurals acquired their cartilaginous shape at around 25 to 30 mm SL, after notochord flexion (Fig. 2, Fig. 4D, Fig. 5J–L). The anterior-most epural, usually in the ural centrum 1 or 2 segment formed first and was followed sequentially by more posterior epurals (Fig. 2, Fig. 5J–L). Mineralization started in the center of the diaphysis of epural 1 between 40 and 50 mm SL and formed a ring that eventually extended towards the epiphyses. The posterior-most epurals were small and often mineralized late in ontogeny as in other gars (Grande, 2010; Schultze and Arratia, 1986). All epurals were, however, completely ossified at around 95 mm SL (~124 mm and 120 dpf, Fig. 1D, Fig. 2, Fig. 6C–D).

In teleosts, epurals can shift position relative to their initial position during development, possibly due to notochord flexion (Doosey and Wiley, 2015). While some authors have named epurals based on their position relative to the ventral caudal fin elements with which they align early in ontogeny (Doosey and Wiley, 2015), in our case, it was exceedingly difficult to consistently apply this naming system to many specimens due to ambiguity in establishing the associations of ural centrum, hypural, and epural, especially after notochord flexion. Thus, to remain consistent between samples, we called the most anterior epural “epural 1” and subsequently increased the count posteriorly.

Fully developed spotted gar are said to have “true epurals” (Grande, 2010; Schultze and Arratia, 1986) because the gar epurals resemble detached neural spines. In contrast, bowfin “epurals” resemble pterygiophores, which are skeletal elements supporting the dorsal and anal fin rays and are positioned between successive neural spines, in between segments (Grande and Bemis, 1998; Schultze and Arratia, 1986). In our samples of spotted gar, epurals differed from neural spines by not being attached to the dorsal side of the vertebrae and by being median to the body axis even if, as mentioned above, a few posterior preural vertebrae displayed median neural spines instead of paired neural spines. Notably, we never observed two epurals associated with the same centrum or neural spine, and we did not observe any epural directly joined to a neural arch or spine. In our specimens, epurals started condensation separated from the neural spines or spine instead of detaching from them, in contrast to previous observations (Schultze and Arratia, 1986; Schultze and Arratia, 1989). In our fish, epurals varied in their separation from their associated neural spine: some epurals seemed to grow between neural spine rudiments, but others remained distant (Fig. 5L, Fig. 6C–D). This finding confirms that gar epurals are most likely to be “true epurals” because of their position and numbers. Their origin and formation by cartilage cell condensation within the mesenchyme, however, appears to be different from the formation of neural spines and, in our samples, epurals did not detach from neural arches but formed separately from them.

Our fully developed spotted gar usually had five epurals, but some had as few as three and others as many as seven (Fig. 6C–D), consistent with numbers reported in gars by others: five in longnose gar (Nybelin, 1977), five to eight in longnose and shortnose gars (Schultze and Arratia, 1986), and four or five in spotted gar and from three to eight in the six other extant gar species (Grande, 2010) (Table 3). The greater range we observed is again likely due to our larger sample size and/or rearing conditions. The number of epurals was positively correlated with the number of hypurals (Pearson’s correlation p-value = 2.98 × 10−5, R2=0.3018) (Fig. 6E). This correlation could be explained by variations in the number of somites composing the ural part of the caudal fin skeleton in some individuals, generating an irregular number of additional centra, hypurals ventrally, and epurals dorsally, as a metameric unit (Doosey and Wiley, 2015). These variations in the number of ural somites are known to result from the loss of regular metamerization in the posterior region of the body in actinopterygians (Schultze and Arratia, 2013; Witten and Hall, 2015).

The dermoskeletal caudal elements: caudal fin rays and fulcra

Caudal fin rays

Juvenile gar in our sample set already had actinotrichia projecting ventrally and dorsally from the notochord before the resolution of the median fin fold into anal, dorsal, and caudal fins (data not shown) as previously reported (Grande, 2010). The dermal formation of the lepidotrichia, which are the mineralized portions of the fin rays (Grande, 2010) formed by two half cylindrical elements (the hemilepidotrichia), initiated in the hypaxial portion of the caudal fin around hypurals 1 and 2, and progressed distally in fin rays located anterior and posterior to hypurals 1 and 2 (Fig. 4B–C). Our finding differs from a previous report that lepidotrichia first form at the boundary between hypurals 2 and 3 (Metscher and Ahlberg, 2001). Rays located anterior to the two initial rays developed before posterior rays, which developed following the formation of the hypural elements with which they associate (Fig. 4C–E, Fig. 5M). Mineralization, as evidenced by Alizarin Red uptake, occurred at around 12 mm SL (~16 mm TL and 15 dpf, Fig. 1D), progressed distally up to 20 mm SL (~28 mm TL and 24 dpf, Fig. 1D), and terminated at around 30 mm SL (~37 mm TL and 28 dpf, Fig. 1D), although rays continued later to thicken, elongate and ossify distally (Fig. 2, Fig. 4B–F). Lepidotrichia were absent dorsal to the notochord in juvenile or adult gar, consistent with previous observations (Grande, 2010; Nybelin, 1977), which may be due to either a secondary loss or their incorporation into the dorsal caudal fringing fulcra (Grande, 2010).

Fully developed spotted gar in our samples generally had 12 principle rays in their caudal fin that were all branched and segmented, and no procurrent rays (small, unsegmented and unbranched rays located along the dorsal and ventral edges of the posterior region of the caudal peduncle). This count is consistent with the 11 to 13 reported previously in other gar species (Grande, 2010; Nybelin, 1977; Schultze and Arratia, 1986) (Table 3). Our spotted gar had fewer rays (12 in general) than they had hypaxial elements (usually 13 or 14), and thus, one or two hypurals did not support a caudal fin ray, consistent with prior studies (Schultze and Arratia, 1989).

Fulcra

Basal fulcra are dermal mineralized elements originating from modified scales, and are found in basally diverging actinopterygians, including acipenseriformes and basal teleosts, but have apparently been lost several times independently in the lineages of extant polypteriformes, bowfin, and teleosts (Arratia, 2009). In our spotted gar specimens, fulcra formed both dorsally and ventrally at the basal edges of the caudal fin (Fig. 1A, Fig. 2, Fig. 4E, Fig. 7). The basal fulcra appeared well before other scales in the caudal region, but still relatively late in ontogeny, with the anterior-most fulcra forming first dorsally above hypurals 1 and 2 around 37 mm SL (~43 mm TL and 33 dpf, Fig. 1D, Fig. 2, Fig. 4E, Fig. 5L), and then, around 55 mm SL (~70 mm TL and 60 dpf, Fig. 1D), ventrally below haemal spine 6 or 7 (Fig. 2, Fig. 4E). This finding is consistent with the first signs of fulcra mineralization at 45 mm TL in longnose gar (Grande, 2010). Fulcra formation was reported to first appear at 241 mm TL in shortnose gar (Schultze and Arratia, 1986), but we think that this report was more likely referring to fringing fulcra (fulfr), which border the leading edges of the marginal caudal fin rays, than to basal fulcra (fub), which are located at the bases of the marginal rays. We confirm that early developing fulcra stained with Alcian Blue for a short period before taking up Alizarin Red (Arratia, 2009) (Fig. 5L). The dorsal anterior-most basal fulcrum had an arrowhead shape (Fig. 7A, fub) and the ventral anterior-most basal fulcrum had a tear-drop shape (Fig. 7D, fub). Because subsequent basal fulcra are divided in two and appeared as pairs across the midline for both dorsal and ventral leading edges of the fin (Fig. 7A, D), spotted gar fulcra are of type ‘Pattern I’ based on Arratia’s classification of fulcra (Arratia, 2009). During development, each new basal fulcra was longer than the previous one as they formed posteriorly and covered the basis of the dorsal and ventral edges of the caudal fin (Fig. 7). These split ‘Pattern I’ basal fulcra are referred to as rudimentary rays by Grande (Grande, 2010) because of their shape, localization and basal articulation like rays, but our developmental studies suggest that basal fulcra have a different origin than rays and that a pair of basal fulcra is not homologous to a pair of hemilepidotrichia forming a rudimentary ray (developing fulcra, fub, in Fig. 5L, and developing caudal fin ray, cfr, in Fig. 4C). Thus, we prefer the term ‘paired basal fulcra’ (Arratia, 2009) even though little difference may exist between rudimentary rays and paired basal fulcra (Schultze and Arratia, 1989). Ventral basal fulcra were usually less numerous and more elongated than dorsal basal fulcra and sometimes displayed spinous ornamentation (Fig. 7D). Some fossil actinopterygians also show ornamentation on basal fulcra (Arratia, 2009). Fringing fulcra, because they form as small paired spiny bony elements lying on the leading edges of the dorsal and ventral marginal principal rays posteriorly to the paired basal fulcra (Fig. 1A, Fig. 7B–C, Fig. 7E–F), are thus of type C (Arratia, 2009; Grande, 2010); fringing fulcra appeared later in development than basal fulcra and became more numerous in larger fish.

Figure 7.

Figure 7

Caudal fulcra in spotted gar. (A) 90 mm SL. (B & E) 416 mm SL. (C & F) 471 mm SL. (D) 98 mm SL. Alcian Blue Alizarin Red cleared and stained dorsal (A) and ventral (D) basal fulcra dissected and flat mounted; Alcian Blue Alizarin Red cleared and stained dorsal (B) and ventral (E) fulcra; demerstid beetle cleaned dorsal (C) and ventral (F) fulcra. Scale bars indicate 1 mm. Abbreviations: cfr, caudal fin rays; fub, unpaired basal fulcra; fufr, fringing fulcra; or, ornamentation; pfub, paired basal fulcra; sca, scales.

Two plates of connective tissue and two types of distal caudal radials

Two plates of connective tissue, separated between hypurals 2 and 3, stained variably with Alcian Blue in many of the smaller specimens (below 65 mm SL, ~85–90 mm TL and 75–85 dpf, Fig. 1D). Stained plates were present only in specimens smaller than 65 mm SL; at the corresponding position, larger fish contained two types of distal caudal radials (Fig. 2, Fig. 8A–C). Little is known about the plates of connective tissue in gars, likely because few studies exist on juvenile gar caudal fins. The plates of connective tissue are generally observed in teleost caudal fins and are thought, together with the distal caudal radials, to support the caudal lepidotrichia (Arratia and Schultze, 1992; Schultze and Arratia, 1988; Schultze and Arratia, 1989).

Figure 8.

Figure 8

The gar caudal fin is organized around two plates of connective tissue. (A) 43 mm SL. (B) 62 mm SL. (C) 184 mm SL. Animals were stained with Alcian Blue and Alizarin Red. Scale bars indicate 1 mm. (D–E) 17 mm SL, cross section; scale bar is 100 μm; (F) 17 mm SL, sagittal section, scale bar is 100 μm; (F′) 17 mm SL, sagittal section, scale bar is 25 μm. D–F are sections stained with Alcian Blue and counter stained with Nuclear Fast Red. The white elongated triangle indicates hypural 1 and the black arrow points at the hypural diastema. Abbreviations: pdcr, post-element distal caudal radials; idcr, intercalated distal caudal radials; hs, haemal spin; hyp, hypural; nc, notochord; pct, plate of connective tissue; pcta, anterior plate of connective tissue; pctp, posterior plate of connective tissue.

The anterior, larger plate of connective tissue first showed Alcian Blue uptake at around 10 mm SL and it surrounded and capped the distal epiphyses of the first two hypurals, the parhypural, and haemal spines 2 through 6 or 7 (Fig. 2, Fig. 8A–B), matching the variability of the number of haemal elements associated with the caudal fin. Histological analysis of young gar caudal fins showed that the plate surrounds the distal epiphysis of the hypaxial elements both laterally and ventrally, and joins each adjacent element. (Fig. 8D–F). The position of the anterior plate of connective tissue is in agreement with a described “core of connective tissue”, or “plate of connective tissue”, early in the ontogeny of the genus Lepisosteus (Schultze and Arratia, 1989). A similar cartilaginous element also appears in a photograph and interpretative drawing of a young specimen of alligator gar, but without any comment (Fig. 281 in (Grande, 2010)).

In addition to this anterior plate of connective tissue, a smaller posterior plate spanned hypurals 3 to 4 or 5 in 20 of the 77 specimens (26%) that had the anterior plate stained with Alcian (Fig. 8A–B). This posterior plate of connective tissue, recalling the dorsal plate of connective tissue in teleosts (Arratia and Schultze, 1992), has, to our knowledge, not been previously described in any species other than in teleosts. Its description here marks a previously unknown feature of gar caudal fin that we later discuss in detail (Desvignes et al., 2017).

In gar of increasing size, the anterior plate of connective tissue became fragmented and took up progressively less Alcian Blue stain, with stain diminishing first between the distal epiphyses, and then along the rest of the structure (Fig. 8A–C). Two events occurred concomitantly with the fading of Alcian uptake: 1) the distal caudal fin radials formed by detaching from the distal portions of the hypaxial elements from which they extended (Fig. 8B–C) and 2) some chondrocytes located between the distal epiphyses of the hypaxial structures associated with the plate began to condense and to increase their uptake of Alcian Blue stain (Fig. 8B–C). Larger specimens, from 65 mm SL and above, while lacking the plates of connective tissue, showed two types of distal radials: 1) radials resulting from the separation of the cartilage previously capping the hypaxial elements (Fig. 8B–C, pdcr), and 2) smaller radials intercalated between the distal epiphyses of the hypurals and haemal spines encompassed by the anterior plate of connective tissue (Fig. 8B–C, idcr). Grande clearly recognized a link between radials and the plate of connective tissue because he labeled the anterior plate of connective tissue in a young specimen of alligator gar “dcrb” for “a bar of cartilage posterior to the hypurals that fragments into distal caudal radials early in ontogeny” (Fig. 281 in (Grande, 2010)). Furthermore, distal radials have been observed at the distal end of hypaxial elements in longnose gar (Nybelin, 1977), depicted in shortnose gar (Fig. 7A in (Schultze and Arratia, 1986)), were described in Florida gar (Fig. 24A in (Bartsch, 1988)), and have been discussed for the genus Lepisosteus and drawn in longnose gar (Fig. 17 in (Schultze and Arratia, 1989) and Fig. 15A in (Lauder and Liem, 1983)), and they were depicted in longnose and alligator gars (Fig. 93–95, 283–286 in (Grande, 2010)), but the explanation here is the first to describe their origin. In addition, distal radials, both at the distal end of hypurals and intercalated between their distal epiphyses, were also present in larger spotted gar specimens at the location of the posterior plate of connective tissue, similar to the distal radials found in place of the anterior plate of connective tissue (Fig. 8C). Radials distal to hypurals 3 and 4 have been depicted in figures of longnose gar (Fig. 1B by (Nybelin, 1977) and Fig. 7A in (Schultze and Arratia, 1986)), of Florida gar (Fig. 24A in (Bartsch, 1988)), and of longnose and alligator gars (Fig. 94–95 and 286 in (Grande, 2010)), but these authors did not call attention to them or discuss their possible significance.

Distal caudal radials are often referred to as cartilage elements present at the distal ends of some haemal spines and hypurals of the caudal fin of many teleosts and non-teleost actinopterygians (Arratia and Schultze, 1992; Grande, 2010; Nybelin, 1977; Schultze and Arratia, 1986). The main distal radials in the spotted gar caudal fin skeleton seemed to form by the separation of the cartilaginous tips of the distal epiphyses of hypaxial elements within the plates of connective tissue, and to remain aligned with the hypural or haemal spine they detached from, as in many teleosts, including basally diverging teleosts (Hiodon, Albula, Elops) (Schultze and Arratia, 1988), salmonids (Arratia and Schultze, 1992) and other teleosts (Schultze and Arratia, 1989). These cartilages are usually referred to as “post-element” cartilages (e.g. post-hypural cartilages) (Fujita, 1989) because they appear to be prolongations of the hypaxial elements they detach from during the fragmentation of the two plates of connective tissue. Concomitantly, the condensation of chondrocytes intercalated between distal epiphyses of the hypurals or haemal spines associated with the plates of connective tissue led to the formation of a second type of smaller distal caudal radials. These cartilaginous elements, associated with the fragmentation of the plate of connective tissue, were previously called “intercalary cartilages” (Cope, 1890) or “accessory cartilages” (Rosen, 1973) or “inter-[element] distal cartilages” (Fujita, 1989) in various teleosts. Because they consistently localized between adjacent hypaxial caudal fin skeletal elements in our gar, we propose to call these radials “intercalated distal caudal radials”. This name seems more informative than “accessory cartilages”, is simpler to use, and appears to be more appropriate than “intercalary”. The consistent localization of the intercalated distal caudal radials suggests that they could develop from localized modifications of cells from the plate of connective tissue similar to the distal caudal radials drawn in longnose gar (Fig. 17 in (Schultze and Arratia, 1989)), and the unnamed irregular cartilaginous elements observed for example in Hiodon (Schultze and Arratia, 1988) and salmonids (Arratia and Schultze, 1992). Notably, in some of our spotted gar individuals, some intercalated cartilages were fused with post-element cartilages (e.g. Fig. 6F). In addition, the consistent presence of intercalated distal caudal radials between hypaxial elements except for hypurals two and three, i.e. the location of the gap between the two plates of connective tissue, reinforces the conclusion that the intercalated distal caudal radials likely arise from the fragmentation of the two plates of connective tissue. To our knowledge, this is the first description of two types of distal caudal radials in any non-teleost actinopterygian (Arratia and Schultze, 1992).

Conclusions

This investigation of the morphology and ontogeny of the spotted gar caudal fin skeleton identified and confirmed similarities in caudal fin skeletal organization among gar species, and showed that gar species differ mostly in the numbers of meristic features. In addition, our study revealed several previously undescribed features of the gar caudal skeleton, including 1) two different types of distal caudal radials that 2) replace two transient plates of connective tissue, thus revealing 3) the organization of the developing spotted gar caudal skeleton into two hypaxial ensembles separated by a space between hypurals 2 and 3. The two plates of connective tissue, the two types of distal caudal radials, and the space between hypurals 2 and 3 are also apparent (but generally not commented on) in previously published figures of other gar species; thus, we conclude that these three features represent anatomical properties of the caudal fin skeleton that were likely present in the last common ancestor of all lepisosteiformes and recall the hypural diastema complex known in Teleostei (Arratia and Schultze, 1992; Desvignes et al., 2017). The identification of previously insufficiently recognized anatomical features in the gar caudal fin skeleton highlight the power and importance of ontogenic studies in providing compelling evidence for the identification of the evolutionary relationships of skeletal elements. Together, this work sets the stage for future evolutionary, morphological, functional, and gene expression studies involving both teleost and non-teleost species that is enabled by the release of the spotted gar reference genome sequence, improved captive rearing, and the development of developmental genetic tools for spotted gar.

Experimental Procedures

Origin of sampled fish

Wild adult spotted gar broodstock was collected by electrofishing from the Atchafalaya River Basin, Louisiana and cultured in a 2 m diameter tank containing artificial spawning substrate under a natural photoperiod. Females were injected with Ovaprim© (0.5 ml/kg) to induce spawning. Fertilized eggs, embryos, and juveniles were reared in 10-gallon tanks, at 24° C, under a 14 h light/10 h dark photoperiod regime. Daily care was performed by the University of Oregon fish facility staff. Animals were handled in accordance with good animal practice as approved by the University of Oregon Institutional Animal Care and Use Committee (Animal Welfare Assurance Number A-3009-01, IACUC protocol 12-02RA).

Skeletonized Gar

One adult specimen of spotted gar (SL=471 mm), wild-caught in Lake Cataouatche in Louisiana, was prepared for a skeletonized view using dermestid beetles (Fig. 1A).

Alcian Blue and Alizarin Red Staining

Following euthanasia with an overdose of MS-222 (Finquel - Argent Labs), fish were fixed in 4% paraformaldehyde (PFA), washed, and serially transferred into 80% ethanol for storage or immediate staining. Fish were stained first with Alcian Blue for cartilage, enzymatically cleared using 1% trypsin, bleached in 3% hydrogen peroxide, differentially stained with Alizarin Red for bone, and finally cleared with increasing solutions of glycerol (Walker and Kimmel, 2007). The length of each step was adjusted according to the size of the fish. Some samples were stained with only Alizarin Red.

In total, 149 spotted gar individuals, ranging from 9 mm to 416 mm standard length (SL) were cleared and stained for this study. Samples ranged from freshly hatched juveniles to wild-caught mature adults several years old (Fig. 1C). Of these 149 fish, 139 were stained for both cartilage (with Alcian Blue) and bone (with Alizarin Red), but because strong cartilage staining can inhibit or hinder the uptake of Alizarin Red in bone (Bird and Mabee, 2003), we stained ten fish only for bone. Indeed, 15 Alcian Blue/Alizarin Red double-stained preparations provided ambiguous details on bone mineralization, so those specimens were scored only for early cartilage stages corresponding to the condensation of cartilage cells and initial Alcian Blue uptake. Figure 1C presents the distribution of sample sizes for various age fish.

Imaging and Scoring

After staining, fish were observed using a Leica M165 FC stereomicroscope and scored using an 8-stage scoring system that ranges from no cartilage cells identifiable to fully ossified bones (Table 1). Images were taken with a Leica DFC425 C camera mounted on the Leica stereomicroscope for small specimens, or alternatively, for larger specimens, with a Canon EOS60D DSLR body mounted with a Canon EF100 mm f/2.8 macro lens. Scoring of all specimens was performed by the same person to optimize consistency. Pearson’s correlation was performed using R (v3.1.2) software.

Table 1.

Scoring system use for developmental descriptions of skeletal elements.

Score Description
0 No cartilaginous cells (chondrocytes) identifiable
1 Condensed cartilaginous cells (chondrocytes), but no Alcian Blue staining
2 Initial uptake of Alcian Blue (faint staining)
3 Significant uptake of Alcian Blue (strong staining), no Alizarin Red staining
4 Initial (faint) Alizarin Red staining
5 Significant Alizarin Red staining in less than half of the entire structure
6 Significant Alizarin Red staining in half or more of the entire structure
7 Entire structure stained with Alizarin Red (ignoring joints, which never mineralize)

Histology

Caudal fin regions of 15–17 mm SL spotted gar larvae (14–15 dpf) were fixed in 4% PFA, embedded in paraffin wax and sectioned at 7μm. Cross sections and sagittal sections were then deparaffinized and stained with Alcian Blue for the presence of cartilage (blue), and nuclei were counter-stained to a red color with nuclear fast red following a slightly modified protocol from Bancroft and Gamble (2008). Sections were then observed and imaged on a Leica DFC310 FX camera mounted on a Leica DMLB binocular microscope.

Terminology

Table 2 summarizes the essential terminology of caudal fin skeletal elements based on definitions from Grande (Grande, 2010), from Schultze and Arratia (Schultze and Arratia, 2013), and from ZFIN and Fishbase (Bradford et al., 2011; Froese and Pauly, 2015); Figure 1A–B illustrates most of these definitions.

Table 2.

Caudal fin skeleton terminology.

Term Symbol Definition
Actinotrichia Slender rods of collagen (i.e. elastoidin) that are the main support of fin-folds in young stages, and around which lepidotrichia later develop, while remaining at the tip of each lepidotrichia in adults.
Basidorsal arcualia darc Small, paired cartilaginous dorsal vertebral elements forming the bases of the neural arch (the neural arch, which ossifies, is an extension of this element) (Fig. 5G–I). Basidorsal arcualia are also called basidorsal cartilage or basidorsals.
Basiventral arcualia bv Small, paired cartilaginous ventral vertebral elements forming the bases of the haemal arch (the haemal arch, which ossifies, is an extension of this element) (Fig. 5F). Basiventral arcualia are also called basiventral cartilage or basiventrals.
Caudal fin skeleton Posterior-most portion of the axial skeleton. The centra of the anterior most caudal fin skeleton support the anterior most haemal element that supports a caudal fin ray (Fig. 1B); has two sub-regions: the preural and ural regions defined by the nature of the centra (Fig. 1B).
Caudal skeleton Portion of the axial skeleton posterior to the abdominal skeleton and anterior to the caudal fin skeleton (Fig. 1B).
Centrum c The central body of each vertebra; represented by mineralized, calcified, or ossified portions that surround the notochord (Fig. 1A).
Distal caudal radials dcr (pdcr and idcr) Small caudal skeletal elements located distal to proximal radials (haemal elements and hypurals) can be of several types, here the “post-element distal caudal radials” (pdcr) and the “intercalated distal caudal radials” (idcr) (Fig. 8C).
Embryonic fin fold ff Median fold of skin surrounding the body, from which the dorsal, anal and caudal fins develop (Fig. 3A–B).
Epaxial elements Dorsal skeletal elements positioned above the notochord/vertebral column (e.g. neural spines and epurals).
Epichordal and hypochordal lobes el and hl Lobes of the caudal fin located dorsal (epi-) or ventral (hypo-) to the notochord (nc) (Fig. 3B).
Epural ep Autonomous neural spine of a preural or ural vertebra that may support fin rays (Fig. 5L).
Fulcra pfub, fub, and fufr Modified scales located at the dorsal and ventral fin margins present only in acipenseriformes and gars among extant species; two types in gars: basal fulcra, present on both the dorsal and ventral bases of the caudal fin, either paired (pfub) or individual (fub), and fringing fulcra (fufr), which border the marginal rays of the caudal fin (definition according to (Arratia, 2009)) (Fig. 7).
Haemal arch ha Ventral extension from a caudal vertebra enclosing the main arteries and veins of the caudal region (Fig. 5F).
Haemal element Compound element formed by a haemal spine and a haemal arch (Fig. 5F).
Haemal spine hs Ventral extension of the haemal arch forming, with the haemal arch, the haemal element (Fig. 5F).
Hypaxial element Skeletal element positioned ventral to the notochord/vertebral column (e.g. haemal elements and hypurals).
Hypural hyp Modified haemal spine that is associated with a ural centrum and has lost its haemal arch and haemal canal; may be articulated or fused with its respective ural centrum (Fig. 3B).
Lepidotrichia or caudal fin rays cfr Bony segmented rods found in fins of bony fishes; develop around actinotrichia as part of the dermal exoskeleton (Fig. 3D–F).
Neural arch na Paired element forming an arch surrounding the neural canal on the dorsal side of a vertebra; develop from the basidorsal arcualia (Fig. 1A).
Neural spine ns Distal extension of a neural arch dorsal to the neural canal; usually paired (nsp), but can be median in the most posterior few preural centra and ural centra (Fig. 1A).
Opisthural lobe ol Fin-like structure protruding from the dorsal base of the caudal fin lobe and lying superior to it in young fishes; contains the notochord; previously called the “notochordal appendage” (Carpenter, 1975). (Fig. 3C–D)
Parapophysis pp Lateral or ventrolateral projections of a vertebra arising by the replacement ossification of basiventrals or direct formation into bone (Fig. 1A).
Parhypural phy Haemal element supported by preural centrum 1 (pu1); its arch marks the exit of the main caudal arteries and veins from the haemal canal to the side of the hypurals. (Fig. 1A)
Plate of connective tissue pct Cartilaginous element occupying the distal portion of some hypaxial elements, thought to support the caudal fin rays together with the distal radials; also called “core of connective tissue”. (Fig. 8A)
Polyural caudal skeleton Type of caudal skeleton characterized by the presence of more than two ural centra.
Preural centrum pu A centrum in the caudal fin region preceding the ural centra, bearing both neural and haemal arches and usually both neural and haemal spines, but no hypural; numbered from the posterior-most element, which supports the parhypural, toward the anterior-most element which supports the anterior-most haemal spine supporting a caudal fin rays. (Fig. 1B)
Principal caudal ray Al the segmented and branched dermoskeletal rods plus usually one unbranched but segmented rod located at each of the dorsal and ventral edges in the fin lobe; associated with endoskeletal elements. (Fig. 3E)
Procurrent caudal ray Dermoskeletal rods, shorter than a principal caudal ray, which form the dorsal and ventral series of lepidotrichia of median fins and are associated with endoskeletal elements, such as pterygiophores, neural and haemal spines, epurals, and uroneurals.
Proximal radials Haemal elements and hypurals in contrast to distal radials that are located distal to haemal elements and hypurals.
Ural centra u Posterior-most centra of the vertebral column characterized by the absence of haemal arches and the support of hypurals ventrally; numbered beginning from anterior to posterior; ural centrum 1 (u1) supports hypural 1 (Fig. 1A–B).
Vertebra A member of a set of all serially repeated, ossified, cartilaginous, and ligamentous elements around the notochord, consisting of centrum, neural arch and spine, and haemal arch and spine (Fig. 1A).

Supplementary Material

Supp FigS1

Supplemental Figure 1. Sequence of development and ossification of individual caudal fin skeletal elements based on average values. Diamonds indicate earliest appearance of elements by cell condensation. Blue indicates Alcian Blue uptake and development of cartilage. The blue-to-red transition represents the progression of ossification. Red represents fully mineralized structures. Less opaque bars refer to elements found in less than 25% of the studied specimens.

Supp FigS2

Supplemental Figure 2. Sequence of development and ossification of individual caudal fin skeletal elements based on the fastest growing individuals. Diamonds indicate earliest appearance of elements by cell condensation. Blue indicates Alcian Blue uptake and development of cartilage. The blue-to-red transition represents the progression of ossification. Red represents fully mineralized structures. Less opaque bars refer to elements found in less than 25% of the studied specimens.

Key findings.

  • Observations presented here provide the most detailed description of the developmental origins of caudal fin skeletal elements in any Holostean species to date.

  • Two types of distal caudal radials replace two transient plates of connective tissue, identifying two hypaxial ensembles separated by a space between hypurals 2 and 3.

  • These novel features have not been described in any gar species, but can be observed in published figures of other gar species, and thus represent anatomical structures common to lepisosteiformes.

  • The present work highlights the power and importance of developmental studies, and provides the basis for future evolutionary, morphological, and functional investigations on a variety of actinopterygians.

Acknowledgments

The authors would like to thank Allyse Ferrara and Quenton Fontenot at Nicholls State University for gar fishing and for setting up laboratory broodstock and laboratory spawns. We thank the University of Oregon Aquatics Facility, Trevor Enright, Andy Kimm, Javier Camoriano, and Tim Mason for help in gar rearing; Bonnie Ullmann, Jamie Nichols, and specially Charles B. Kimmel and Gloria Arratia for insightful comments, and two anonymous reviewers for comments that improved the clarity of the manuscript. The work was supported by NIH grant 5R01 OD011116 (JHP).

Grant Sponsor: This work was funded by the grant NIH 5R01 OD011116 (JHP).

Footnotes

Authors’ contributions

Study concept and design: T.D. and J.H.P. Fish sampling: T.D., A. C., I. B., and T. E. Acquisition of data: T.D., A.C., and T.E. Analysis and interpretation of data: T.D., A.C., T.E., and J.H.P. Drafting of the manuscript: T.D. and A.C. Critical revision of the manuscript: T.D., A.C, I.B, T.E., and J.H.P. Statistical analysis: T.D. and A.C. Obtained funding R01 OD011116 J.H.P. Study supervision: T.D. and J.H.P.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigS1

Supplemental Figure 1. Sequence of development and ossification of individual caudal fin skeletal elements based on average values. Diamonds indicate earliest appearance of elements by cell condensation. Blue indicates Alcian Blue uptake and development of cartilage. The blue-to-red transition represents the progression of ossification. Red represents fully mineralized structures. Less opaque bars refer to elements found in less than 25% of the studied specimens.

Supp FigS2

Supplemental Figure 2. Sequence of development and ossification of individual caudal fin skeletal elements based on the fastest growing individuals. Diamonds indicate earliest appearance of elements by cell condensation. Blue indicates Alcian Blue uptake and development of cartilage. The blue-to-red transition represents the progression of ossification. Red represents fully mineralized structures. Less opaque bars refer to elements found in less than 25% of the studied specimens.