Skeletal ontogeny of the Plainfin Midshipman, Porichthys notatus (Percomorphacea: Batrachoidiformes)

Abstract Batrachoidiformes are benthic fishes that utilize the undersides of rocks as spawning nests. Their larvae are attached to the nest and nourished by a large yolk sac. The evolutionary shift from feeding, free‐swimming larvae to sedentary larvae that are reliant on their yolk sac for nutrition can lead to changes in skeletal development. Batrachoidiformes also have many morphological specializations, such as five pectoral‐fin radials (versus four in other acanthomorphs) that are of uncertain homology, the determination of which may have phylogenetic implications. A larval series of Porichthys notatus was collected and its skeletal ontogeny is described. In P. notatus the ossification of the pharyngeal toothplates occurs relatively later than in percomorphs with free‐swimming larvae. The posterior basibranchial copula cartilage (= fourth basibranchial) in Porichthys notatus has a unique development among fishes: it initially develops as a paired element at 6.8–7.1 mm NL before fusing posteriorly and forming single median cartilage at 7.4 mm SL. Cartilages of hypobranchial four are transitory, being observed in two specimens of 6.8 and 7.3 mm NL before fusing with ceratobranchial four. The previously identified dorsalmost pectoral radial is a bone formed by a hypertrophied propterygium that ossifies later in development. The earliest stages of P. notatus have three dorsal spines, but during late larval development, the growth of the third dorsal spine is interrupted. The development of P. notatus is compared and discussed in context to that of other acanthomorph.


| INTRODUC TI ON
Ontogenetic variation represents a fundamental source of morphological variation, together with individual and phylogenetic variations (Grande, 2004;Hilton & Bemis, 2012). Early ontogeny in particular is often useful in comparative anatomical studies of fishes and other organisms to inform hypotheses of homology for unique anatomical structures. For example, Johnson and Britz (2005) demonstrated that the clavus of Molidae (Tetraodontiformes) is formed by elements of the posterior ends of both dorsal and anal fins, refuting previous hypothesis that the clavus was a highly modified caudal skeleton. Ontogenetic variation is also informative in systematic analysis. Hilton et al. (2019) demonstrated that the pattern of ossification of the vertebral column within species of Bathymasteridae (Zoarcoidei) is variable and suggests that the distinct pattern observed in Ronquilus and Bathymaster could be a synapomorphy grouping these taxa. As for any morphological feature, the systematic information of ontogenetic studies of a particular group of organisms requires broadly comparative data.
These fishes are commonly known as toadfishes and midshipman.
These are small to medium-sized fishes (total length, TL, ranging from 20 to 50 cm), and have a wide, stocky body with a dorsoventrally compressed head that bears fleshy supraorbital and oral cirri (Greenfield et al., 2008). Despite the relatively small size of this order, Batrachoidiformes are distributed worldwide, inhabiting mostly benthic habitats of coastal regions (Collette, 2005;Greenfield et al., 2008). Reproductive behavior and early life history are unknown for most species of Batrachoidiformes. Collette (2005) summarized most of the information known for the order, and the most detailed accounts comes from Poricthys notatus (Arora, 1948), Aphos porosus (Balbontín et al., 2018), Opsanus tau (Dovel, 1960), and, a lesser extent, Halobratrachus didactylus (Felix et al., 2016).
Male toadfishes are nest builders and vocalize to attract females during the spawning season (Arora, 1948;Balbontín et al., 2018;Dovel, 1960;Felix et al., 2016;Rice & Bass, 2009). Females lay large eggs (>5 mm diameter) on the roof of nests that are formed by rocks or other hard substrates (Arora, 1948;Britz & Toledo-Piza, 2012;Dovel, 1960). After hatching from the egg, larvae remain attached to the nest while gradually absorbing their yolk sac. The time that larvae are attached to the nest is dependent on water temperature and it may take up to 60 days for a larva to detach from the nest and become a free-swimming juvenile (Balbontín et al., 2018).
Despite extensive descriptions of the external morphology in early development of Opsanus tau and Porichthys notatus (Arora, 1948;Dovel, 1960;Watson, 1996), studies and descriptions of the development of the internal morphology from early-life to adult stages are lacking for Batrachoidiformes. Balbontín et al. (2018) offered a generalized skeletal description of larval specimens of Aphos porosus, however, they did not provide accounts of individual bones and cartilages. Felix et al. (2016) focused their ontogenetic descriptions on the stato-acoustic organs and swimbladder of larval stages of Halobatrachus didactylus.
The goal of this manuscript is to describe the ontogeny of the skeleton in Porichthys notatus, the Plainfin Midshipman. This species is endemic to the northeast Pacific Ocean, occurring in coastal areas from British Columbia (Canada) to Bahia Magdalena (Mexico; Walker & Rosenblatt, 1988). These descriptions are then used as the basis for the assessment of homology for specializations of the batrachoidiforms, which contributes to a better understanding of the diversity and evolution of highly modified morphological structures of the order.

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VAZ and HILTON given structure present. The terms "minimum length or size" or "earliest occurrence" refer to the smallest size at which a cartilage or a bone was found. Lengths are indicated as notochord length (NL) for specimens without caudal-fin rays or standard length (SL) when caudal-fin rays are developed. Terminology of skeletal elements follows Hilton (2011) and (for the caudal skeleton) Vaz and Hilton (2020). Terminology of early cartilages of the neurocranium follows de Beer (1937). Porichthys notatus (as do Batrachoidiformes in general; Collette, 2005) has an extended yolk-sac larval stage and lacks a free-swimming larval stage. The term larva as used here follows Richards (2005) and was used for specimens that hatched from the egg but remained attached to the rock in the nest, whether or not the yolk-sac was externally visible or internalized (although still present; Figure 2). The term juvenile was used for specimens that were detached from the rock, but were free-swimming in their nests. Among the specimens examined, the smallest free-swimming specimens of Porichthys notatus were 24 mm SL.
The neurocranium of Porichthys notatus gradually becomes more dorsoventrally flattened during ontogeny. The width-to-depth ratio is approximately 1:1 to from 6 to 10 mm SL. At larger sizes this ratio increases to approximately 1.5:1 at 13.1 mm SL, and to 2:1 when larvae are free swimming (24.3 mm SL).

| Orbital region
The smallest specimen examined (VIMS 40287, 5.4 mm NL) has a pair of elongate cartilages, the trabecula cranii, that extend the length of the anteromedial ventral surface of the head (Figure 3a).
Distinct trabecula cranii cartilages with a similar arrangement are observed to 18.6 mm SL (Figure 3h). Although most of the trabecula cranii cartilages are replaced by the basisphenoid in larger larval specimens, remains of these cartilages can be observed in both larva ( Figure 3i) and juveniles (VIMS 38018). The taenia marginalis cartilage is first observed in the posterodorsal region of the orbital region at 6.0 mm NL and is fixed at 7.7 mm SL (Figure 3c).  Figure 3g).

Frontals
Frontals are first observed at 7.1 mm NL, fixed at 8.7 mm SL, and are located along the lateral margin of the dorsal surface of the head, dorsomedial to the anterior edge of the taenia marginalis ( Figure 3d).

Parasphenoid
The parasphenoid is first observed at 6.0 mm NL, fixed at 7.5 mm SL, as a triangular, laminar dermal bone. It is positioned in the middle of the hypophysial fenestra (i.e., the space between the cartilages of trabecula cranii). At 7.9 mm SL, the parasphenoid extends from the anterior third to the middle of the ventral surface of the neurocranium and extends posteriorly to the level of the anterior margin of the sagitta, acquiring an arrow shape (Figure 3c). At 9.7 mm SL, the parasphenoid develops a slender ascending process, extending posterodorsally from its posterior third (Figure 3e). At 11.8 mm SL, the parasphenoid extends posteriorly, nearly contacting the anterior margin of the basioccipital. The ascending process of the parasphenoid is rectangular and contacts the prootic at 18.6 mm SL (Figure 3h), and covers its anteroventral edge at 24.3 mm SL (Figure 3i). At this size the tip of the anterodorsal process of the autosphenotic becomes rounded and forms an acute angle with the lateral margin of the frontal, similar to that observed in larger juveniles (Figures 3j,k).

Basisphenoid
The earliest stages of ossification of the basisphenoid occur in the posterodorsal margin of the trabecula cranii at 11.8 mm SL and are

Pterosphenoid
The taenia marginalis cartilages are entirely replaced by bone at 18.6 mm SL, forming a triangular pterosphenoid (Figure 3h). The dorsal margin of the pterosphenoid is straight and attaches to the ventral margin of the frontals, with its posterodorsal edge contacting the dorsal region of the prootic (Figure 3i). The triangular outline of the pterosphenoid remains in juveniles, but in those specimens the ventral edge of the pterosphenoid extends farther ventrally, contacting the dorsal margin of the ascending process of the parasphenoid.

| Otic region
The anterior basicapsular commissure is visible at 6.0 mm NL ( Figure 3a). This commissure is located in the posterior extremity of the trabecula cranii and projects dorsolaterally, continuous with the auditory capsule. Anterior to the commissure there is a small triangular postpalatine process. At 6.2 mm NL, the postpalatine and prootic processes meet to form the lateral commissure, enclosing the foramen for passage of the trigeminal nerve (as seen in Figure 3b).
The auditory capsule is a rectangular cartilage that extends laterally at the level of the sagitta. The anteroventral edge of the auditory capsule bears the prootic process. The posteroventral edge of the auditory capsule also has a small projection that represents the early stages of the posterior basicapsular commissure (Figure 3a

Prootic
The prootic first appears at 8.7 mm SL and fixes at 11.8 mm SL. The

Exoccipital
The exoccipital first appears at 7 mm SL (fixed at 7.9 mm SL) within

| Jaws and hyopalatine arch
The sequence of ossification of jaws and hyopalatine arch is: dentary (fixed length at 6.8 mm NL; minimum length observed is 6.0 mm NL), angular and maxilla (fixed, 6.9 mm NL; minimum, 6.0 mm NL), pre- is a triangular lamina at 6.2 mm NL ( Figure 5b) but by 7.1 mm NL the palatoquadrate projects substantially anteriorly and the anteriormost point (i.e., the pars autopalatina) is slightly expanded. At 7.4 mm SL (Figure 5d), the pars autopalatina projects slightly dorsally, and at 7.5 mm SL, the anterior tip of the pars autopalatina start projecting anteriorly; this projection forms a distinct cartilaginous process at 9.7 mm SL ( Figure 5g). The hyosympletic cartilage is present and already has a distinct foramen for passage of the hyomandibular branch of the fascialis nerve in the smallest specimen observed (5.4 mm NL). The pars hyomandibularis is trapezoidal and bears a posterior process for articulating with the opercle (Figures 5a-e). The pars symplectica is rod-shaped, tapering anteroventrally, extending close to the jaw joint (Figures 5a-f).

Dentary
The dentary first develops at 6.0 mm NL and is fixed at 6.8 mm NL.

Mentomeckelian
Ossification of the mentomeckelian first appears at 8.7 mm SL and is fixed at 10.9 mm SL as an endochondral ossification near the anterior tip of Meckel's cartilage (as seen in Figure 5h). The mentomeckelian fuses to the dentary by 16.1 mm SL (Figure 5i).

Anguloarticular
The angular first appears at 6.0 mm NL and is fixed at 6.9 mm SL. In

Retroarticular
The retroarticular is first observed at the posterior tip of Meckel's cartilage at 7 mm SL and fixed at 8.2 mm SL. This bone becomes triangular by 16.2 mm SL, reflecting the condition observed in juveniles. At 24.3 mm SL, this triangular-shaped bone is tightly attached to the posterior margin of the anguloarticular bone.

Quadrate
The quadrate is first present at 8.7 mm SL, fixed at 9.7 mm SL

Maxilla
The maxilla was first present in a specimen of 6.0 mm NL but is fixed only at 6.9 mm NL. At its earliest occurrence, the maxilla is a small, thin, and laminar bone and is smaller than the interhyal.

Premaxilla
The premaxilla first appears at 7 mm SL, fixed at 7.7 mm SL as a small yet elongate bone (less than one third the length of the maxilla), with a slightly enlarged anterior tip (

Rostral cartilage
The rostral cartilage is first observed at 8.8 mm SL and is fixed only at 11.8 mm SL. In its earliest stages, the rostral cartilage is approxi-

Opercle
The opercle first appears at 6.0 mm NL as an inverted L-shaped bone with a small socket at its anterodorsal corner to meet the opercular process of the hyosymplectic cartilage (Figure 5a). At 6. and juveniles.

Subopercle
The subopercle first appears at 6.0 mm NL and fixes at 6.2 mm NL; ( Figure 5b). In the earliest sizes, this bone is concave, narrow, and as long as the opercle, and begins to expand anteriorly at 6.8 mm NL (Figure 5c). At 7.1 mm NL, it acquires a comma shape, with its anterior edge as wide as the opercle and its posterior region ta-

Interopercle
The first occurrence of the interopercle is at 6.0 mm NL, but it is only fixed at 6.8 mm NL. When it first appears the interopercle is a short thin bone (approximately one-third of subopercular length) that is positioned laterally to the ventral tip of the interhyal (Figure 5c).
At 7.1 mm NL, the interopercle expands dorsoventrally and is approximately one-third of the length of the interhyal. It continues to elongate, and at 7.5 mm SL it is more than one-half the length of the subopercle. At larger sizes, most of the growth of the interopercle occurs in the dorsoventral direction, and by 11.8 mm SL ( Figure 5h) the interopercle is as long as the interhyal and has the trapezoidal shape that is seen in larger larvae and juveniles (Figures 5i,j).

Preopercle
The preopercle is the last bone of the opercular series to become fixed, 7.5 mm SL, although its first occurrence is also at 6.

| Ventral hyoid arch
The

Interhyal
The smallest specimen with the earliest stages of ossification of the interhyal has 8.7 mm SL, but ossification of this bone fixes only at 10 mm SL. The interhyal becomes fully ossified (except the dorsal and ventral tips) at 11.8 mm SL (Figure 5h). At this size, the interhyal already has the shape seen in larger larvae and juveniles: a small,

Posterior ceratohyal
The first trace of bone of the posterior ceratohyal appears at 9.7 mm SL and is located slightly anterior to its articulation with the interhyal (Figure 8c). At 10.9 mm SL, the posterior ceratohyal

Branchiostegals
Each individual branchiostegal ossifies in a proximal-to-distal direction, but as a series they develop in a posterior to anterior direction.
The two most posterior branchiostegals are present at 6.0 mm NL, and by 6.4 mm NL, the two central branchiostegals appear (Figure 8a).

Urohyal
The urohyal first appears at 8.7 mm SL and is fixed at 11.8 mm SL. In the smallest sizes, the urohyal is a small, pyramid-shaped bone with its base directed anteriorly and in close contact with the ventral surface of the copula communis (Figure 10e). The urohyal expands laterally and resembles two half-moons fused medially at 11.8 mm SL (Figure 10f). By 16.1 mm SL, the medial portion of the urohyal thickens and projects ventrally, forming a T-shaped bone that is similar in form to that of juveniles and adults (Figure 10g,h). The sequence of development of the ventral hyoid arch is summarized in Figure 9.
3.6.1 | Ceratobranchial series A 6.0 mm NL specimen has the four anterior ceratobranchial cartilages (Figure 10a). At this size, the three anterior cartilages are elongate and similar in size; ceratobranchial 4 is approximately one-half the length of ceratobranchial 3; by 6.2 mm NL it is approximately three-fourths the length of ceratobranchial 3 and by 7.9 mm SL it is similar in length to the other ceratobranchials, as seen in adults. The fifth ceratobranchial cartilage has a fixed occurrence in specimens larger than 6.6 mm NL (Figure 10b), but this cartilage is present in one specimen with 6.0 mm NL (VIMS 42847). In the smallest sizes, the ceratobranchial 5 is a small circular cartilage that grows to ap- Ceratobranchial 4 has a single row of six gill rakers. Although more distinctly formed in larger larval sizes, their shape remains pyramidal and unicuspid, lacking the multiple cusps that are observed on the gill rakers of juveniles and adults.

| Hypobranchial series
Hypobranchial cartilages 1 and 2 are already present at 6.0 mm NL (Figure 10a), and until 6.4 mm NL, they are concave. From 6.8 mm NL to 9.7 mm SL (Figures 10b-d), these cartilages are straight, and from 10.9 mm SL (Figure 10e), they are slightly convex, as seen in adults.
Hypobranchials 3 appear to be present in a 6.0 mm NL specimen, but their presence is only fixed at 7 mm NL (Figure 10b). In the earliest stages, hypobranchials 3 are two pairs of circular cartilages that are in series with hypobranchials 1 and 2 (Figure 10b). Hypobranchial 3 acquires a semi-circular shape by 7.4 mm SL (Figure 10c), and by 9.7 mm SL it develops an anteroventrally directed process. A pair of cartilages between the anteromedial edge of ceratobranchial four and posterior copula was observed in two specimens (VIMS 40274, 6.8 mm NL; VIMS 40283, 7.3 mm NL; Figure 10b). Considering their position, these cartilages are tentatively identified as hypobranchial four. In other teleosts hypobranchial four cartilages are transitory and fuse to ceratobranchial cartilages during development (e.g., Salmo, de Beer, 1937; Salminus brasiliensis, Mattox et al., 2014; in the latter, inferred by their Figure 10, although this element was not labeled). Additional specimens are needed to confirm whether the observed pair of cartilages are indeed hypobranchial four that fuse to cerabranchials during development or if they are malformations in these two specimens.

Hypobranchials
The earliest perichondral ossification of hypobranchials 1 and 2 is = at 8.7 mm SL, but it becomes fixed at 10.9 mm SL. By 11.8 mm SL

| Basibranchial series
The copula communis is a rod-like cartilage that extends from the hypohyals to the level of the third ceratobranchials in the smallest size observed (6.0 mm NL) to 16.2 mm SL (Figures 10a-g).  (Figures 10c,d). The resulting median element is heart-shaped at 7.4 mm SL to 8.2 mm SL, before becoming V-shaped at larger sizes. The posterior copula (=basibranchial four) remains cartilaginous in juveniles and adults.

Basibranchials
Two ossifications of the copula communis (basibranchials 1 and 3) are found in juveniles and adults. The earliest trace of ossification of basibranchial 3 appears at 13.9 mm SL and is fixed at 16.1 mm SL (Figure 10g). At 18.6 mm SL ( Figure 10h) the basibranchial three has replaced most of the posterior region of the copula communis cartilage, forming a rectangular bone that is similar to the shape observed in larger larvae and juveniles. An ossified basibranchial 1 was not found in any larval specimens, but in juveniles (VIMS 38018, 84.5 mm SL) it is formed by a small circular bone that is close to the anterior tip of the copula communis.

| Epibranchial series
The earliest stages of epibranchials 1-4 are rectangular cartilages that are first observed at 6.4 mm NL and fixed at 7.4 mm NL (Figure 10c).

Upper pharyngeal tooth plates
The upper pharyngeal toothplates first appear on the third pharyngobranchial at 11.4 mm SL (Figure 10f), although a pair of small pharyngeal teeth was associated with pharyngobranchial three at 8.2 mm SL. At 16.1 mm SL, the pharyngeal toothplate three already covers most of the ventral surface of pharyngobranchial three. The pharyngeal toothplate 2 is first observed at 13.9 mm SL and its occurrence is fixed in specimens larger than 16.1 mm SL. In its earliest stages, the pharyngeal toothplate two is restricted to the posterolateral edge of the second pharyngobranchial (Figure 10g). At 16.1 mm SL, the teeth that will attach to the upper pharyngeal toothplates are already present, but they have not ankylosed to the toothplate yet (as observed in larger larvae and juveniles; Figure 10h). The earliest trace of the toothplate of epibranchial three is observed on the ventromedial region of epibranchial three at 18.6 mm SL, at which point there is only a single tooth present (Figure 10h). At 24.3 mm SL, the toothplate of epibranchial three still only has a single tooth, but the toothplate is already projecting laterally and has the initial stages of development of a second tooth. The sequence of development of gill arches is summarized in Figure 11.

| Vertebral column and intermuscular bones
The sequence of ossification of the vertebral column and intermuscular bones is: neural arches (first observed and fixed at 6.0 mm NL), haemal arches (fixed length at 6.4 mm NL; minimum length observed at 6.0 mm NL), epineural bone one (fixed,  (Figures 12-14).
Only the neural and haemal elements of the third and second preural centra and the basiventrals of vertebrae 2-5 are pre-formed in cartilage.   (7.9 mm SL) already has fully ossified centra from the vertebrae 1-31.

Neural arches and spines
The smallest specimen to have all 42 preural vertebrae with ossified centra is 10.9 mm SL (Figure 13c). From this and larger sizes (including juveniles), the vertebral column comprises 11 abdominal vertebrae, 31 caudal vertebrae, and two ural vertebrae.

Intermuscular bones
The first bone of the intermuscular series to appear is the anteriormost epineural bone, present in a 7 mm SL specimen but fixed only

| Caudal fin and skeleton
Development of the skeleton of the caudal fin and skeleton of Porichthys notatus is described and discussed in Vaz and Hilton (2020); an overview is provided here but the reader is directed to Vaz and Hilton (2020) for details on the ontogeny of this complex. The sequence of ossification of the caudal-fin skeleton occurs in the following order: principal fin rays (fixed length at 7.9 mm SL; minimum observed length at 7 mm SL); compound ural centrum one, ural centrum two, and neural arch of second preural centrum (fixed, 9.7 mm SL; minimum, 8.7 mm SL); dorsal hypural, ventral hypural, procurrent fin rays, and haemal arch of second preural centrum (fixed, 10 mm SL; minimum, 8.7 mm SL); parhypural (fixed, 11.5 mm SL; minimum, 8.7 mm SL); haemal spine of second preural centrum (minimum and fixed at 13.1 mm SL); anterior and posterior epurals (fixed, 13.9 mm SL; minimum, 8.7 mm SL); and neural spine of second preural centrum (fixed, 16.1 mm SL; minimum, 13.9 mm SL) (Figures 15, 16).
The first element of the caudal skeleton to appear is the cartilage of the ventral hypural, fixed at 6.4 mm NL, followed by the dorsal hypural cartilage, fixed at 7.1 mm NL, and the parhypural cartilage, fixed at 7.5 mm SL (although the smallest occurrence of all these cartilages is 6.0 mm NL, VIMS 42847; Figures 15a-c). The parhypural and ventral hypural develop from a single group of cartilage cells.
The arch of the parhypural first develops between 6.4-6.8 mm NL, but it is not until 7.3 mm NL-7.9 mm SL that the spine of the parhypural becomes distinct (Figures 15d-f). Two epurals are present in Porichthys notatus, with both cartilages first appearing at 7.9 mm SL; its occurrence is fixed by 8.7 mm SL (Figures 15g-i). The ventral arcocentrum of preural centrum two is present between 6.7 mm NL-7.8 mm SL (Figures 15e,f). A cartilaginous haemal spine of preural centrum two is first present at 7.5 mm SL but is fixed only after 8.7 mm SL (Figure 15g-i). The dorsal arcocentrum of preural centrum two is first observed at 7 mm SL, but its occurrence is fixed only at 9 mm SL (Figure 15h). The cartilage of neural spine of preural centrum two first occurs at 8.7 mm SL (Figures 15h,i).

Hypurals and Parhypural
Ossification of the dorsal and ventral hypural is fixed at 10 mm SL (Figures 15j,k), whereas for the parhypural it is fixed at 11.4 mm SL (Figures 15m,n). The smallest occurrence of ossification of these structures, however, all occur at 8.7 mm SL. At 18.6 mm SL, these elements are fully ossified (Figure 15r). Fusion of ventral and dorsal hypurals to their associated ural centra (first and second ural centra, respectively) is first observed by 14.3 mm SL (Figure 15o). The ossifications of the parhypural and ventral hypural fuse by 11.8 mm SL (Figure 15m).

Epurals
Ossification of epurals is first observed at 8.7 mm SL, but fixed only at 13.9 mm SL (Figures 15n-q). Complete ossification of epurals occur at 18.6 mm SL (Figure 15r).

Ural centra
The first and second ural autocentra ossify at 8.7 mm SL (fixed length at 9.7 mm SL; Figure 15i). Ossification of both ural central is complete at 11.9 mm SL (Figures 15m).

Preural centrum two
Ossification of the ventral arcocentrum of preural centrum two is first observed at 8.67 mm SL (and fixed at 10 mm SL; Figure 15i).
Ossification of haemal spine of preural centrum two occurs at 13.1 mm SL. Ossification of neural arch of preural centrum two is first observed at 8.7 mm SL (fixed at 9.7 mm SL). Ossification of the cartilage of neural spine of preural centrum two first occurs at 13.9 mm SL, but is only fixed at 16.1 mm SL (Figure 15q).

Caudal-fin rays
The first fin rays to appear are the innermost pair of the principal series that delimit the diastema at 7 mm SL, but their occurrence are fixed at 7.9 mm SL (Figures 15d-f). Ossification of fin rays runs from the diastema to both dorsal and ventral margins. The principal series (I, 6, 6, I) are complete at 10.0 mm SL (Figure 15j-r). At this size, both ventral procurrent fin rays and the second dorsal procurrent ray are present (Figure 15j).

| Anal fin and support
The sequence of ossification of the anal fin is: anal-fin rays (fixed length at 7.9 mm SL; minimum length observed at 7 mm SL), anal-fin proximal radials (minimum and fixed at 11.8 mm SL), and anal-fin middle radial (minimum and fixed at 13.9 mm SL) (Figures 17-19).
Proximal-middle radial cartilages of the anal fin are first seen at 6.0 mm NL and have their occurrence fixed at 6.9 mm NL. A 6.

Proximal radial
Ossification of the proximal radials occurs at 11.8 mm SL in the first seven anterior pterygiophores of the anal fin (Figure 17a).

F I G U R E 1 6
Diagram of sequence of development of the caudal skeleton of Porichthys notatus. Bars correspond to fixed length. Error bars correspond to the smallest size that a structure was found. Bars in blue correspond to cartilage, red to bone. Lengths presented in mm NL/SL.

Middle radial
First ossification of the middle radial of anal fin occurs at 13.1 mm SL. At 14.3 mm SL, ossification of the middle radial is observed on pterygiophores 11 to 17 (Figure 17b). All but the four most anterior and two most posterior middle anal-fin radials are ossified at 16.1 mm SL. At 18.6 mm SL, the four anterior middle radials remain cartilaginous (Figure 17d). At this size, the middle radials of the anal fin are rectangular and laterally compressed. In juveniles, the middle radial become spool-shaped.

Distal radial
In all larval specimens, distal radials remain rounded and cartilaginous. In juveniles, the center of the anal-fin radials ossify, although their external surfaces are cartilaginous where they contact the bases of the anal-fin rays (Figure 18a).

Anal-fin rays
The first soft fin rays of the anal fin to develop are the five anterior rays (smallest size observed at 7 mm SL; fixed at 7.9 mm SL). In the earliest stages, the hemitrichia are small (less than a third of the length of the associated proximal-middle radial cartilages). All more posterior anal-fin rays are present by 11.8 mm SL (Figure 17a), although the hemitrichia are unsegmented and unbranched and still have actinotrichia exposed at the tip of the most posterior fin rays; the length of these fin rays is similar to that of the proximal-middle radial cartilages. Segmentation and branching of anal-fin rays are first observed at 14.3 mm SL (Figure 17b). At 18.6 mm SL (Figure 17d), all anal-fin rays are segmented and branched, their length are approximately two to three times longer than the length of their supporting pterygiophore.

| Dorsal fins and supports
The sequence of ossification of the dorsal fin is: dorsal-fin rays and middle radials are fully fused (i.e., they are proximal-middle radials). The third proximal-middle radial is also a single elongate bone, although it lacks the cartilaginous tips found in the more anterior proximal-middle radials. At 24.5 mm SL, this proximal-middle radial is still distinct but the third dorsal-fin spine is smaller than a distal radial cartilage. In large juveniles (84.8 mm SL; Figure 18d), the third proximal-middle radial is no longer than the base of the second dorsal-fin spine.

Middle radial
Ossification of middle radials of the second dorsal fin 11-28 is observed at 16.1 mm SL (Figure 17c). By 18.6 mm SL, ossification of the middle radials are observed in all but the four most anterior soft-fin ray pterygiophores and in the two most posterior radials (Figure 17d). At this size, the middle radials are rectangular and laterally compressed. In juveniles, the middle radials acquire a spool shape.

Dorsal-fin rays
The smallest specimen with soft dorsal-fin rays is 7.7 mm SL, but their occurrence is fixed only at 8.7 mm SL. In the earliest stages, hemitrichia are small (length less than one-third that of proximal-middle cartilages) and are associated with proximal-middle cartilages 5-14 ( Figure 17A). Soft rays and spines of the dorsal fin develop in both anterior and posterior directions. The posteriormost soft dorsal-fin ray develops at 13.7 mm SL. At this size, most dorsal hemitrichia are still unsegmented and unbranched but are as long or longer than the proximal-middle radial cartilages. At 18.6 mm SL (Figure 17d), all dorsal-fin rays are segmented and branched, their length are approximately two to three times longer than the length of their supporting pterygiophore.

Dorsal-fin spines
Three dorsal-fin spines are present in the earliest sizes of development of Porichthys notatus studied (Figure 18b). The third dorsal-fin
The scapulocoracoid cartilage is rectangular and the scapular fenestra is present at its first appearance (6.0 mm NL). At 6.2 mm NL (Figure 20a), the coracoid process is distinct and projects antero- to 11.5 mm SL, the propterygium and pectoral radials are all connected distally.

F I G U R E 19
Diagram of sequence of development of dorsal and anal fins of Porichthys notatus. Bars correspond to fixed length. Error bars correspond to the smallest size that a structure was found. Bars in blue correspond to cartilage, red to bone. Lengths presented in mm NL/SL.

Cleithrum
At the smallest size examined (5.4 mm NL) the cleithrum is present as a slender rod of bone, extending from the base of the occipital region of the neurocranium to the ventral margin of the abdominal cavity (Figure 20a). At 7.1 mm NL, the cleithrum is slightly expanded laterally (Figure 20b), and by 7.9 mm SL its ventral tip is projected anteriorly (Figures 20d,e). By 16.1 mm SL, the cleithrum attains its adult form (Figure 20f). The posterodorsal process of the cleithrum first appears at 7.5 mm SL (Figure 20c) as a slender process. It gradually widens until achieving a trapezoidal shape at 16.1 mm SL, which is similar to its form in adults (Figure 20f).

Supracleithrum
The smallest specimen with a supracleithrum is 6.0 mm NL and it is fixed at 6.2 mm NL. In its earliest stages, the supracleithrum is a slender bone that is less than 25% the width of the cleithrum

Scapula
The scapula is first observed at 13.9 mm SL but its occurrence is fixed only at 18.6 mm SL (Figure 20f). The scapula is mostly rectangular with a convex posterior margin in all larvae, similar to the shape the observed in juveniles. The scapula is pierced by the scapular fenestra ( Figure 20f).

Coracoid
The first indication of ossification of the coracoid is at 13.1 mm SL.
The coracoid has a trapezoidal outline and its ventral margin bears an anteroventrally directed process. Ossification of this process is incomplete even in adults; the tip of the coracoid process remains cartilaginous (Figure 20f).

Propterygium and pectoral radials
Complete separation among all pectoral radials and the propterygium first occurs at 11.8 mm SL (Figure 20d). Ossification of pectoral radials starts in the middle of the cartilage at 10.9 mm SL and progresses both anteriorly and posteriorly. The propterygium ossifies in a similar manner, but ossification is first observed at 11.8 mm SL. In larger larvae and juveniles, the propterygium is a large pad-

Pectoral-fin rays
The pectoral-fin rays are first found in a 6.0 mm NL specimen, but the ossification of the rays is not fixed until 7.4 mm SL. The first rays to appear are located at the dorsal margin of the propterygium (Figure 20c). The other fin rays develop in a ventral direction gradually at later sizes. By 18.6 mm SL, there are 18 pectoral-fin rays present; 21 fin rays are present in juveniles. Segmentation of the fin rays is first observed at 11.8 mm SL. Between 11.8 and 16.1 mm SL, the pectoral-fin rays develop a triangular dorsal projection on the base of the medial hemitrichium; this projection contacts the adjacent hemitrichium. Distal radial cartilages are first observed at 7.9 mm SL at the tips of the two dorsal pectoral radials. The series of distal radials gradually develops both dorsally and ventrally. A small, unsegmented pectoral-fin ray element that we interpret as a pectoral-fin spine articulates with the posterodorsal edge of the propterygium and projects dorsally (Figure 20d-f). Fusion of the halves of the pectoral-fin spine occurs after 10.9 mm SL.

| Pelvic girdle and fin
The sequence of ossification of the pelvic fin is: pelvic-fin rays

Pelvic-fin rays and spine
The pelvic fin comprises one spine and two fin rays. Fin rays and the pelvic-fin spine are first seen at 6.0 mm NL, but their occurrences are fixed at 7 mm SL (rays) and 7.9 mm SL (spine).
By 10.9 mm SL, the length of the pelvic-fin rays is twice that of the spine (Figure 22a). By 11.8 mm SL, the pelvic-fin rays become segmented (Figure 22b). From this size through the juvenile stages, the pelvic-fin rays are three to four times longer than the pelvic-fin spine (Figure 22c,d).

| DISCUSS ION
The description of the early skeletal development confirms that Porichthys notatus lacks the mesethmoid and parietal bones in all stages of ontogeny. The mesethmoid and parietal are not found in any other species of Batrachoidiformes examined to date. Assuming Porichthys notatus as an exemplar of the order, this study offers additional support for the absence of these bones as synapomorphies of Batrachoidiformes (Wiley & Johnson, 2010).
This dataset demonstrated that Porichthys notatus lacks the intercalar throughout its ontogeny. The intercalar is present in most species of Halophryninae (except Halobatrachus and Colletteichthys).
All representatives of Batrachoidinae, Porichthyinae, and Thalassophryninae observed to date lack an intercalar. Preliminary analysis suggests that the absence of the intercalar is a potential synapomorphy grouping these three subfamilies, being lost homoplastically in Halobatrachus and Colletteichthys (Vaz, 2020 to basibranchial one and a small circular element that is ventral to the posterior copula cartilage (Figure 23d).

| Developmental comparisons to other percomorphs and teleostean fishes
There is a broad range of data available related to the early development of the skeleton of fishes, many of which have been fundamental to hypotheses of homology for various structures within Actinopterygii (e.g., Arratia & Schultze, 1990, 1991, 1992. Several of these studies are focused on particular anatomical regions, such as skull (de Beer, 1937), jaws, and hyopalatine arch (e.g., Arratia, 1990, Siluriformes;Arratia & Schultze, 1991, basal Actinopterygii;Konstantinidis & Johnson, 2012b, Tetraodontiformes), caudal skeleton Konstantinidis & Johnson, 2012a). The series of papers by Potthoff (1974Potthoff ( , 1975Potthoff ( , 1980Potthoff and Kelly, 1982;Potthoff & Tellock, 1993;Potthoff et al., 1980Potthoff et al., , 1984Potthoff et al., , 1988 bone of the neurocranium to ossify after the pterotic and is followed by the basisphenoid, prootic, and supraoccipital. In Sciaenops ocellatus, the prootic develops after the frontal, followed by supraoccipital, vomer, pterotic (these three at same size), followed by the epioccipital, then later the autosphenotic. The pterosphenoid is the last bone of the neurocranium to ossify in P. notatus, whereas in S. ocellatus the last bone to appear is the basisphenoid.
Within the suspensorium, jaws, and opercular series, the opercle is the first bone to form in Porichthys notatus, followed by the subopercle, and then the interopercle, which appears at the same time of the dentary. After these elements, the angular, maxilla, and the preopercle appear. In Sciaenops, the maxilla is the first bone to develop, followed by the dentary, opercle, and premaxilla. Following the ossification of the preopercle, the sequence of ossification in P. notatus is premaxilla, articular, retroarticular, hyomandibular, symplectic, and the quadrate.
In contrast, in S. ocellatus the bones that ossify before the preopercle are the anguloarticular, symplectic, endopterygoid, quadrate, and subopercle; after the preopercle is present, the subopercle, hyomandibula, retroarticular, and ectopterygoid appear. It is notable that in P. notatus the endopterygoid is one of the last bones to ossify in the entire skeleton, while in S. ocellatus it develops relatively early.

Because of the phylogenetic distance between Porichthys and
Sciaenops, as well as the complexity of the variation involved in the sequence of ossification, these differences are difficult to interpret, particularly having multiple bones of the skull being coopted to multiple functions (e.g., oral jaws acting in both breathing and feeding).
However, it is possible to make a few generalizations from the developmental data for P. notatus presented above compared to that of S.
ocellatus and other teleosts.
In Porichthys notatus bones that are closely associated with breathing (e.g., opercular series and branchiostegals) ossify at smaller sizes (i.e., earlier stages) than those that are exclusively associated with feeding (e.g., tooth plates and tooth patches of the pharyngeal jaws; Figure 24); the pharyngeal tooth plates are the last elements to develop among elements of the visceral arches in P. notatus. This is possibly related to endogenous feeding through absorption of the yolk during the larval development of P. notatus (Arora, 1948;this study). This is in contrast to S. ocellatus, a species that begins exogenous feeding in their earliest stages (FAO, 2022); in S. ocellatus tooth plates and ceratobranchials among the earliest elements to ossify (Kubicek & Conway, 2016

| Homology of pectoral-fin radials and phylogenetic implications
Batrachoidiformes are historically described as having five pectoral fin radials (Günther, 1861;p. 168, "five carpal bones distinctly developed"), in contrast to the condition observed in most Teleostei, which typically have four pectoral radials. Monod (1960) described the dorsalmost pectoral radial of Halobatrachus didactylus (named as "R1") being as long as the other radials, but remaining cartilaginous; Gunther (1861) described it as rudimentary. Greenfield et al. (2008) (Greenfield et al., 2008; this study, Figure 25). Batrachoidinae, Thalassophryninae, Porichthyinae, and the remaining Halophryninae all have a hypertrophied propterygium that is completely or partially ossified (the latter condition observed only in Batrichthys apiatus and Riekertia ellisi). The phylogenetic analysis of Greenfield et al. (2008) resulted in optimizing the cartilaginous state as apomorphic (character [5]: Upper accessory pectoral-fin cartilage: 0 = ossified; 1 = not ossified). Greenfield et al. (2008) coded this character in outgroups with a question mark because their study did not have ontogenetic data to establish the homology of the propterygium. The propterygium of other fishes ossifies during early ontogeny ossifies and fuses with the upper hemitrichium (Kubicek, 2022;Kubicek & Conway, 2016;Marinho, 2022;Mattox et al., 2014), including Sciaenidae, the family of fishes phylogenetic closer to Batrachoidiformes with ontogenetic information on the ossification of the propterygium (sensu Betancur-R et al., 2017). Based on our ontogenetic data, we infer that an ossified propterygium is the plesiomorphic state and a cartilaginous propterygium would be considered apomorphic. The distribution of this apomorphic state of character could support the hypothesis that Triathalassothia, Halobatrachus, Perulibatrachus, and Austrobatrachus form a monophyletic group. The preliminary phylogenetic analysis from Vaz (2020) did not recover these four species forming a monophyletic group. The phylogenetic implications of this character, however, are still under investigation.
Outside of Batrachoidiformes, the only other teleostean fish reported to have five elongate radial or radial-like elements supporting the pectoral fin is Gigantactis longicirra (Lophiiformes; Waterman, 1948;Rosen and Patterson, 1969). There is no developmental evidence to determine whether the cartilaginous radial-like element of Gigantactis is homologous to the propterygium or if it is an additional pectoral radial. Even if the elongated cartilage of Gigantactis is homologous to the propterygium as in Batrachoidiformes, the currently accepted phylogenetic relationships of toadfishes and anglerfishes within Percomorphacea (Betancur-R et al., 2013;Betancur-R et al., 2017) suggest that these are independent (i.e., homoplastic) occurrences.

| Reabsorption of the third dorsal-fin spine during development
Representatives of Porichthyinae and Thalassophryninae have been historically described having two dorsal-fin spines, in contrast to the three observed in Batrachoidinae and Halophryninae (Collette, 1966(Collette, , 1973Gilbert, 1968;Greenfield et al., 2008;Günther, 1861;Walker & Rosenblatt, 1988). The number of dorsal-fin spines has been used for identification purposes and proposing phylogenetic relationships. Our study clearly shows that there are three distinct dorsal-fin spines in the early development of Porichthys notatus ( Figure 18). The third dorsal-fin spine develops similarly to other spines until approximately 13 mm SL ( Figure 18b). Its development is then interrupted (first observed at 16.1 mm SL; Figure 18c), resulting in a rod-like pterygiophore with a diminutive spine at 24.5 mm SL (Figure 18d). In juveniles, the pterygiophore is further reduced to a small, comma-shaped structure that is closely associated with the pterygiophore of the second dorsal-fin spine (Figure 18e).
A reduced third dorsal-fin spine in Porichthys notatus is clearly seen in ontogeny and suggests the need for reevaluation of the condition in other Batrachoidiformes. A similar structure was found in other species of Porichthyinae, as well as in Thalassophryninae; both subfamilies have been described as having only two dorsal-fin spines (Collette, 1966(Collette, , 1973Gilbert, 1968;Greenfield et al., 2008;Walker & Rosenblatt, 1988). In Thalassophryninae, however, the only structure left is a small ossified spine (a reduced pterygiophore is not present). In Daector dowi, Thalassophryne amazonica, and T. nattereri this spine is circular and small, but in T. maculosa this element is elongate, similar to other pterygiophores supporting soft fin rays dorsal-fin spines: 0=no; 1=yes". By examination of their character matrix, the states "no" and "yes" meant the total number of complete dorsal-fin spines in juveniles and adults, as all taxa coded with "yes" are those from Porichthyinae and Thalassophryninae (taxa with two complete dorsal spines) and those coded with "no" have three dorsal-fin spines (Batrachoidinae and Halophryninae). In addition to the evidence of all Batrachoidiformes having three dorsal-fin spines at some stage of their life, the codification of that character itself has problems. Following Sereno's (2007) terminology and methodology for character construction, the character statement proposed by Greenfield et al. (2008) is not an independent variable.
All Batrachoidiformes have at least two fully developed dorsal-fin spines and pterygiophores. The variation relates to the presence of a third dorsal-fin spine. Therefore, incorporating the ontogenetic data from this study, the correct character statement is the development of the third dorsal-fin spine in juveniles and adults. The exclusive conditions (i.e., states of character) would be "complete with fully developed spine" versus "reduced with spine restricted to a circular or elongate bone"

| Development of the vertebral column
In Porichthys notatus the dorsal arcocentra develop in an anteroposterior direction, whereas the ventral arcocentra first appear in the midpoint of the body (between vertebrae 12 to 23) and develop in both anterior and posterior directions. In most percomorph fishes for which developmental information is available, the direction of development of dorsal and ventral arcocentra typically are similar (e.g., in Sciaenidae, Kubicek & Conway, 2016;most Zoarcoidei, Hilton et al., 2019;and Lutjanus, Potthoff et al., 1988), regardless whether the dorsal arcocentra and ventral arcocentra develop anterior to posterior or from the midpoint to the extremities. Potthoff (1975)

| CON CLUS IONS
The propterygium of Porichthys notatus is hypertrophied and ossified, elongating during development to become as long as the pectoral radials. Considering the similarities observed in the pectoral fin across Batrachoidiformes, we infer that a similar developmental pathway occurs in other species of toadfishes. Therefore, we propose that the dorsal most element of the pectoral fin, previously thought to be a pectoral radial, is a hypertrophied propterygium.
Porichthys notatus has three complete dorsal-fin spines in its early life history, and the development of the third dorsal-fin spine interrupted, resulting in a spine that is reduced to a small bone in juveniles. The recognition of this reduced third spine allowed the identification of this reduced spine in other species of subfamilies Porichthyinae and Thalassophryninae, both of which were previously thought to have only two dorsal-fin spines. Beyond the implications of homology, the discovery of a reduced third dorsal spine in these subfamilies leads to changes in their diagnoses.
Instead of describing these subfamilies as having only two dorsalfin spines, the correct characteristic is having a reduced third dorsal-fin spine.