External Morphology of Lophiosilurus alexandri Steindachner, 1876 during Early Stages of Development, and Its Implications for the Evolution of Pseudopimelodidae (Siluriformes)

Pseudopimelodidae are Neotropical catfishes characterized by having slightly to strongly depressed body in fully developed specimens. The largest species of the family with 500 mm SL, Lophiosilurus alexandri, experiences impressive changes in body shape during development, becoming extremely depressed when fully developed. Accordingly, Lophiosilurus alexandri is an ideal species to observe the morphological changes during ontogeny, and to seek solid interpretations on the polarity of characters. Specimens of distinct larval periods (yolk sac, flexion and postflexion; n = 186 specimens) and juvenile stages (n = 20) were analyzed. Changes in body shape, position of mouth and eye, morphology of fins and pigmentation were observed during the development of Lophiosilurus. Larvae (5.7–11.2 mm standard length) had pigmentation concentrated on the head and parts of body, eyes small and pigmented, short barbels, and well-developed finfold. Juveniles (15.9–28.1 mm standard length) had body shape similar to adult, with head depressed and bearing bony ridges, large mouth, dorsally-oriented eyes, small barbels and well-developed shoulder bulges (cleithral width). The greatest morphological changes in the development of L. alexandri occurred during the postflexion larval stage. Relative to standard length, measurements of snout length, head depth and body depth are smaller in juveniles than in larvae, but body width is larger. New interpretations on the phylogenetic characters related to these changes are provided in view of the two alternative hypotheses of the evolution of Pseudopimelodidae.


Introduction
The search for the morphological changes that gave rise to the present diversity of forms and taxa continues to be one of the main goals of Evolutionary Biology. Many of these changes can in fact be interpreted as series of transformations of character states with genetic basis and functional expression. Nevertheless, the phylogenetic significance of these transformation series is unknown for most species of Neotropical fishes. One way to trace a transformation series is to study the ontogeny of a particular species [1]. The Biogenetic law, proposed by Haeckel [2] and reformulated by Nelson [1], says that the steps of an ontogenetic series can be directly translated into an ordered series of character states. That concept, however, has not been accepted among systematists [3]. The main arguments against Nelson's Biogenetic law are that there is no evidence that certain body parts are more conservative or contain more information than other parts, and that the ontogenetic changes permit direct interpretations as ordered character states [4]. Besides these restrictions, Wayne [5] noted that the morphological diversity of adult forms of dogs and other domesticated animals depends on expressions during ontogeny. According to Lovejoy et al. [6], only a confident understanding of the phylogenetic relationships of a group can recover the polarization of characters.
The "pacamã", Lophiosilurus alexandri, is a particularly interesting case for addressing this problem. Lophiosilurus alexandri is the largest species of the family Pseudopimelodidae, reaching 500 mm standard length [7] and 4 kg body mass [8]. This monotypic genus also exhibits the most highly modified morphology for the family. Its head is extremely depressed, wider than long, the eyes are dorsal, and the mouth is wide and prognathous [7]. In the phylogeny of Pseudopimelodidae proposed by Shibatta [9], L. alexandri is in a derived clade, sister to Cephalosilurus. Birindelli & Shibatta [10] corroborated the sister group relationship between Lophiosilurus and Cephalosilurus, but supported that clade sister to all other Pseudopimelodidae based on swim bladder musculature, a hypothesis that corroborated the findings of Ortega-Lara & Lehmann [11]. Therefore, given its large size, unique shape, and controversial phylogenetic position, L. alexandri is a potentially informative species for understanding morphological evolution in Pseudopimelodidae.
Endemic to the São Francisco River basin [12] in northeastern Brazil, Lophiosilurus alexandri is non-migratory and its reproduction involves the construction of a circular nest depression (40-50 cm diameter and 8-10 cm depth) in a shoal area. Spawning is parceled and the female lays large adhesive eggs (3.1 to 3.6 mm) in the center of the nest, which are then guarded and maintained by the male [13]. Upon hatching, the larvae remain at the bottom of the nest and do not move vertically into the water column. According to Tenório [12], it is among the most prized fishes in regional markets, but rarely captured in the upper São Francisco River basin. Due to its excellent flavor and the consistency of its flesh [8], the species has good potential for intensive aquaculture. Every year, juvenile Lophiosilurus alexandri are released into the São Francisco from a hatchery associated with UHE Três Marias as a compensatory measure for construction of the hydroelectric power plant.
Previous studies regarding the anatomy of Lophiosilurus alexandri included Nakatani et al. [14], who described its external morphology from egg to juvenile, and Guimarães-Cruz et al. [15], who described the internal anatomy of L. alexandri based on histological sections of the nervous and digestive systems. The present study contributes with details on the early development of the external morphology in L. alexandri, and new interpretations of phylogenetic characters previously employed by two alternative hypotheses on the evolution of Pseudopimelodidae.

Material and Methods
Developmental series of Lophiosilurus alexandri were obtained from Estação de Piscicultura de Três Marias, CODEVASF, on the upper São Francisco River (Minas Gerais State). Individuals were reared from natural spawning of wild adults in flowing water. Specimens were collected between August 1994 and January 1996, fixed in 4% formaldehyde buffered with calcium carbonate and deposited in the fish collection of the Museu de Zoologia da Universidade Estadual de Londrina (MZUEL), Brazil.
The larval period was divided into three stages (yolk sac, flexion and postflexion) based on the degree of flexion of notochord and development of fins and their supporting elements using standards developed by Ahlstrom & Ball [16], and Kendall et al. [17] modified by Nakatani et al., [14]. Juveniles were determined by the complete development of fins and segmentation of fins rays [14]. A total of 206 specimens were analyzed, including 186 larvae from hatching to 42 days of development (24 yolk sac, 3 flexion and 159 postflexion larvae) and 20 juveniles (49 to 60 days) ( Table 1).
For each stage of development, specimen illustrations were made with the aid of a camera lucida coupled to a stereomicroscope. Multiple illustrations were made for stages that exhibited sudden changes in morphology. Twenty-three measurements were obtained on juveniles using a digital caliper accurate to 0.01 mm, and on larvae using an ocular micrometer attached to a stereoscopic microscope: anal-fin base length (AFBL), body depth (BD), body width (BW), caudal peduncle depth (CPD), caudal peduncle length (CPL), dorsal-fin base length (DFBL), dorsal-fin spine length (DSL), eye diameter (ED), head depth (HD), head length (HL), inner mentonian barbel length (IMBL), interorbital width (IW), interpelvic width (IPW), maxillary barbel length (MXBL), outer mentonian barbel length (OMBL), preanal length (PAL), predorsal length (PDL), prepectoral length (PPL), pectoral-fin spine length (PSL), prepelvic length (PVL), pelvic-fin ray length (PVRL), standard length (SL), and snout length (SnL), Morphometric variables were expressed as percentages of standard length, except for subunits of the head, expressed as percentages of head length. Measurements of width and depth of body and head were taken on juveniles and adults of other species of Pseudopimelodidae (see list of comparative material) for comparison. Meristic data included the number of rays of the pectoral, pelvic, dorsal and anal fins, and total preanal and postanal myomeres. Correlation analyses were carried out using Microsoft Excel, and box plot analysis using Past v. 2.11 [18]. Institutional abbreviations follow Sabaj Pérez [19].
Body elongate, transparent, without dark pigmentation. Yolk sac very large immediately after hatching (Fig 1a), decreasing in volume in specimens one or two days post-hatching (Fig  1b). Notochord straight, visible as transparency for entire length.
Eyes small, not pigmented in newly hatched specimens, but pigmented in one day old specimens. Nostrils undeveloped. Small gill opening present within the first day.
Mouth not formed soon after hatching; visible in subterminal position after the first day of development. Buds of maxillary and mentonian barbels present only within the 1 st day of development. From the second day of development, barbels present. Otic capsule with one semicircular canal visible, and two otholits. Intestine short, anus closed. Primordial fin (finfold) well developed, hyaline, circling the body from the post-cephalic region to the post yolk sac region.
Preflexion. Stage absent in L. alexandri since after termination of the yolk sac stage hypurals present. Large yolk sac persistent on day 4 (Fig 2a and 2b), much reduced at day 9, and completely consumed within the 13 th day of development. In the early stage of postflexion, head deep, with rounded shape in lateral view; depression of head and body starting on day 18. Dorsal region of skull with several ridges on day 19. Mentonian and maxillary barbels larger than in flexion-stage specimens. Outer mentonian barbels and maxillary barbels do not exceed the gill opening. Inner mentonian barbel exceeds posterior margin of eye in early stage of postflexion. Gill opening and nostrils formed. Mouth formed and relatively wide (Figs 3b and 4b); subterminal position persistent until day 17 (Fig 3a), becoming superior after day 27 (Fig 4a). Exogenous feeding is possible after day 3 when the mouth opens.
Primordial fin well developed in early stage of postflexion, surrounding the entire perimeter of trunk and tail; restricted to posterior regions of adipose and anal fins after day 18. Primordial fin further reduced by day 42, but still confluent with caudal fin. Adipose fin defined after 42 days. Dorsal fin distinct in the early stage of postflexion, and fully developed after day 12. Anal fin well developed on day 8, but still associated with primordial fin; final development of anal fin on day 20.
Pectoral fin formation starting on day 7 with emergence of rays; completely developed after day 15. First ray of pectoral fin begins to differentiate on day 6, with small hooks evident along posterior margin; after day 9, hooks visible on both sides with posterior hooks larger than anterior ones (Fig 4c). At 42 days, hooks on anterior and posterior margins of pectoral-fin spine similar in size (Fig 5c). Cleithral bulge and posterior cleithral process weakly developed on day 6, and well developed by the end of postflexion stage. Spurious ray [20] present at the end of pectoral-fin spine since early in larval development (9 days), and persist in adults. Pelvic-fin bud rounded, base large at the start of postflexion stage (day 4). Rays evident on day 4; well developed on day 10. Incipient caudal fin formed by finfold and confluent with dorsal and ventral portions of finfold; dorsal caudal-fin lobe more developed and slightly longer than ventral lobe, with 15 incipient rays visible in 4-day old specimens (Fig 2a). Distal portion of hypural bones, supporting principal rays of caudal fin [21,22], emerge as cartilage on day 3 (flexion stage), and begin to ossify on day 13.
In the early stage of postflexion, dark dendritic chromatophores more concentrated on head, dorsal and ventral regions of trunk from nuchal region to tail, and dorsal region of yolk sac (Fig 2a and 2b). Eyes pigmented. In the late stage of postflexion, chromatophores appear more widespread on body, and more concentrated on head, caudal peduncle, and dorsal-fin base (Fig 4a and 4b).
On day 17 (Fig 3a, 3b and 3c), head longer than wide. Dorsal fin well developed, positioned distinctly in front of middle of body. Axillary gland visible above base of pectoral fin. Pectoral fin, when adpressed, reaching vertical through end of dorsal-fin base and slightly surpassing origin of pelvic fin. Pelvic fin, when adpressed, reaching origin of anal-fin base. Anal fin fully formed, last ray separated from ventral finfold. Primordial adipose fin persistent as part of dorsal portion of finfold, confluent with caudal fin; terminus of adipose fin evident as small notch, otherwise confluent with caudal fin. Caudal fin dorsally confluent with adipose fin; ventral finfold with reduced depth, but connected to anal and caudal fins. Caudal fin with seven dorsal procurrent rays, 13 principal rays and seven ventral procurrent rays (Fig 3a). Dorsal lobe of caudal fin slightly longer than ventral lobe. All fin rays segmented.
At the end of postflexion (42 days ; Fig 4a, 4b and 4c), head width increases, equals length. Skin of head with small wart-like appendages mainly on supraorbital and ventral regions. Dorsal fin remains positioned anterior to middle of body, distant from head by about half the head length. Pectoral fin, when adpressed, reaching vertical through base of fourth ray of dorsal fin, but not reaching pelvic fin. Origin of pelvic fin at vertical through end of dorsal-fin base; pelvic fin reaching anal fin when adpressed. Adipose fin completely separate from dorsal fin, but with small tissue connection to caudal fin. Anal-fin base shorter than adipose-fin base. Dorsal lobe of caudal fin long, almost twice as long as ventral lobe. Caudal fin with 14 dorsal procurrent rays, 13 principal rays, and 12 ventral procurrent rays (Fig 4a).
Juvenile period. Stage observed from 49 to 60 days after hatching. Standard length 15.9 to 28.1 mm. Eyes small relative to head (Table 1). Body and head distinctly depressed (Fig 5a); head approximates hexagonal shape in dorsal view (Fig 5b), wider than long; area of frontal bone depressed, concave in coronal section with conspicuous bony ridges. Mouth large, with prognathic position (Fig 5a) similar to adult specimens (Fig 6a and 6b). Maxillary barbel not reaching gill opening. Tip of outer mentonian barbel surpassing tip of maxillary barbel. Tip of inner mentonian barbel not surpassing posterior margin of eye (Fig 5a). Pectoral girdle well developed, particularly cleithral bulge and posterior cleithral process. Pectoral fin with strong anterior spine and seven soft rays; spine thickened by retrorse hooks on anterior and posterior margins (Fig 5c). Dorsal fin well developed with short, thick, strong anterior spine and six soft rays. Adipose-fin base elongate in specimens at 49, 52 and 56 days post-hatching. Anal fin with 12 unbranched rays. Pelvic fin with 6 rays, third and fourth rays branched in specimens of 52 days or older, fifth and sixth rays branched in specimens of 49 days or older. Dark dendritic chromatophores widespread on head, most concentrated on area of frontal bone; similar chromatophores concentrated on trunk beneath dorsal fin and on caudal peduncle. Base of dorsal fin with chromatophores concentrated in patches. Adipose fin with chromatophores concentrated along base forming a bar that reaches middle of body. Caudal, pectoral and pelvic fins largely hyaline, with sparse dark chromatophores (Fig 5a).
In 60 day old specimens, head wider than long. Dorsal fin in front of middle of body, when adpressed not reaching adipose fin. Skin of head and body with small appendages most evident as bony ridges. Adipose-fin reduced in size with shorter base, separated from caudal fin. Pectoral fin, when adpressed, reaching vertical through dorsal-fin spine, but not reaching pelvic fin. Pelvic fin, when adpressed, no longer reaching anal fin. Anal-fin base becoming longer than adipose-fin base. Caudal fin free from adipose and anal fins, dorsal lobe remains distinctly longer than ventral lobe. Caudal fin with 20 dorsal procurrent rays, 13 principal rays, and 18 ventral procurrent rays. Lateral-line canal well developed to vertical through terminus of dorsal-fin base; lateral line pores evident to vertical through middle of adipose-fin base. Axillary pore very difficult to visualize in larval specimens; associated gland visible by transparency of skin. Only one pore in each side of the body along the surface of the skin under postcleithral process in adult specimens.

Morphometric analysis
Standard length gradually increasing immediately after hatching to nine days post-hatching; relatively constant from 9 to 30 days post-hatching, then increasing more sharply from 30 to 60 days (Fig 7). Body proportions that increase from post-larval to juvenile stages are: eye diameter, head length, dorsal-fin base length, preanal length, prepectoral length, prepelvic length, predorsal length, caudal-peduncle depth, body width, interpelvic width, pectoral-fin spine length, dorsalfin spine length, and pelvic-fin ray length (Table 1). On the other hand, the relative depths of head and body decrease from post-larval to juvenile stages, the same happens to snout length, anal-fin base length, caudal-peduncle length, interorbital width, inner mentonian barbel length, outer mentonian barbel length, and maxillary barbel length (Table 1).
Correlation analysis of body proportions related to body width and depth of larvae and juveniles of Lophiosilurus alexandri yielded a large dispersion, but with a tendency of increase the width over depth, as indicated by the regression line (Fig 8). The inclusion of the width and depth mean proportions obtained from juveniles and adults of other species of the family, shows a wide distribution of these species along the X axis (body depth), but with a tendency to a narrower distribution on the Y axis (body width). Even though almost all species fit the variation of depth and width present in larvae and juveniles of Lophiosilurus alexandri, three species showed values outside of the aforementioned variation: Cruciglanis pacifici has the highest proportion of body depth indicating its relatively deep body, and Microglanis parahybae and M. poecilus have the lowest proportion of body depth indicating its relatively depressed body. The highest value in Fig 8 was obtained by an adult specimen of Lophiosilurus alexandri, due to its distinctly depressed body, more depressed than any other species of Pseudopimedidae.

Discussion
In the development of Lophiosilurus alexandri, major transformations in external morphology were observed between larval stages and between larvae and juveniles. In studies with Hoplias malabaricus and Pyrrhulina australis, respectively, Bialetzki et al. [23] and Taguti et al. [24] observed similar transformations in external morphology during early development. The present observations also corroborate the Theory of Saltatory Ontogeny [25], since development alternated through periods of slow and small changes and fast and large changes. For example, the plateau in standard length during the postflexion stage in L. alexandri (i.e., day 9 to 30) may be explained by the allocation of energy resources in the formation of anatomical structures.
Changes in body shape, position of mouth and eyes, and fin morphology were observed during the development of Lophiosilurus alexandri. Many of these transitional features are seen as distinct terminal conditions in the other species of Pseudopimelodidae, and have been used as characters in phylogenetic analyses. There are some competing hypotheses of the evolution of Pseudopimelodidae based on the morphological characters proposed by Shibatta [9], Ortega-Lara & Lehman [11] and Birindelli & Shibatta [10]. The interpretation of some of these characters based on their ontogeny instead of their terminal condition in fully developed specimens may add clues to the investigation of the pseudopimelodid evolution.
The relative width and depth of body present significant changes during the development of Lophiosilurus alexandri. In fully developed specimens of Lophiosilurus, the body is extremely wide and depressed [26], a condition that is unique among pseudopimelodids and therefore autapomorphic. The extreme condition in L. alexandri is related to its psammophilous and piscivorous behaviors. Lophiosilurus is a bottom-up predator that lurks just below the substrate, leaving only the eyes and mouth exposed as it awaits its prey. The highly depressed body shape is also convergent in some well-known species of benthic fish marine such as Lophius spp. (Actinopterygii, Lophiidae) and Squatina spp. (Chondrichthyes, Squatinidae). However, the condition in larvae and juveniles of Lophiosilurus is similar to the one present in the other species of pseudopimelodids. Therefore, the relative depth of the body is a phylogenetic character with at least two states (perhaps more, if a more accurate morphometric analysis is performed), a relatively deep body being plesiomorphic and a relatively depressed body being apomorphic. This interpretation favors the hypothesis that Cruciglanis and Pseudopimelodus are sister to all other Pseudopimelodidae, both taxa having a relatively deep body (compared to other species of the family). The more shallow bodied Cephalosilurus and Lophiosilurus are thereby derived and nested within the family.
The position of the mouth has also undergone major changes during the development of Lophiosilurus. The mouth is subterminal at the beginning of the yolk sac larval stage. However, the mouth shifts to a terminal position during the end of the yolk sac and early postflexion stages, and eventually becomes superior, prognathous by the end of the postflexion stage, a condition retained into adulthood. This developmental change can also be interpreted as an ordered series of character states. Among adult pseudopimelodids, the putative plesiomorphic state, the subterminal mouth, is present only in Cruciglanis pacifici. An intermediate state, the terminal mouth, is present in adult Batrochoglanis and some Microglanis, sister genera according to Shibatta [9] and Ortega-Lara & Lehmann [11]. A third state, mouth slightly superior, is present in adult Pseudopimelodus and some Microglanis. The most apomorphic state, mouth superior and prognathous, is present only in adult Lophiosilurus and Cephalosilurus, corroborating the phylogenetic hypothesis that these two genera form a clade nested within Pseudopimelodidae [9,11,10]. Ortega-Lara & Lehmann [11] and Birindelli & Shibatta [10] placed Lophiosilurus plus Cephalosilurus as sister to all other Pseudopimelodidae.
Eyes placed laterally on the head are a condition shared by all Pseudopimelodidae except for juvenile and adult Lophiosilurus. Placement of the eyes on the dorsal surface of the head is a condition unique to juvenile and adult Lophiosilurus, and therefore an autapomorphy of the species.
The maxillary barbel is relatively short in Lophiosilurus, falling shy of the dorsalmost portion of the gill opening in specimens of all ages. In fact, during the development, the length of the maxillary barbel decreases compared to the length of head, showing negative allometric growth. The maxillary barbel is also short in all other species of Pseudopimelodidae. Actually, the short maxillary barbel resulting from negative allometric growth seems to be a synapomorphy for the entire family, since long maxillary barbels are present in Heptapteridae and Pimelodidae, the two catfish families most closely related to Pseudopimelodidae [27]. Conversely, the maxillary barbel in Heptapteridae and Pimelodidae show positive allometric growth [14].
The caudal fin in Lophiosilurus is formed and replaces part of the finfold during the postflexion stage. Initially the dorsalmost rays of the caudal-fin upper lobe are longest than the other rays. However, as specimens grow, the dorsalmost rays of the upper lobe become relatively shorter, and the middle rays of the upper lobe are relatively longer, including the adults. The dorsalmost rays of the upper caudal-fin lobe are also longest in larvae of Batrochoglanis raninus [28], indicating that this condition may be also present in the remaining Pseudopimelodidae. Unlike Lophiosilurus, as specimens of Batrochoglanis raninus grow, the upper lobe becomes similar in size to the lower lobe. Based on this ontogenetic series it is possible to interpret this character (shape of the caudal fin) as having three distinct states. In the plesiomorphic state, the dorsalmost rays of the caudal fin are elongate. In the first apomorphic state, the upper and lower caudal-fin rays are approximately similar in length, and the second apomorphic state, the middle rays of the upper caudal-fin lobe become elongated. There is no indication that this character represents a direct (i.e., ordered) transformation series, because the three states are not present in the developmental series of any species. The most plesiomorphic condition is possibly present in the larvae of all species of Pseudopimelodidae, and in fully developed specimens of Cephalosilurus albomarginatus and Microglanis iheringi. The presence of this plesiomorphic state in the latter two species can be interpreted as two independent events of paedomorphosis. On the other hand, the condition of elongate middle rays of the upper caudal-fin lobe is only present in Lophiosilurus, and therefore, another autapomorphy of the species. The caudal fin is responsible for swimming in most fishes [29], and the upper lobe is distinctly wider than the lower lobe in species that are typically benthic and need fast bottomup movements [30,31,32]. Therefore, the shape of the caudal fin in Lophiosilurus is possibly related with its habits as a psammophilous bottom-up predator. The change from the first to the second apomorphic state of this character is also observed in developmental series of many species of Auchenipteridae, Heptapteridae, and Pimelodidae [14,28], indicating that this may be a evolutionary change that occurred early in the evolution of catfishes.
The pectoral-fin spine of fully developed specimens of Lophiosilurus has strong serrations of similar size on the anterior and posterior faces. These serrations are absent in yolk sac and flexion stage larvae, and began to develop in larvae of postflexion stage. The serrations are initially much larger on the posterior face of the spine (Fig 3C), and eventually become similar in size (Fig 4C). Strong serrations of similar size on the anterior and posterior faces of the pectoral-fin spine are present in most species of Pseudopimelodidae. The only exceptions are Pseudopimelodus and Cruciglanis, which have serrations much larger on the posterior face of the spine. The presence of this plesiomorphic state in the latter two genera can be interpreted as an event of paedomorphosis, corroborating Birindelli & Shibatta [10] for considering them as sister taxa.
The lateral-line system is well developed in adults of Lophiosilurus, distributed over the head and as a midlateral canal over the body from head to caudal-fin base. The cephalic portion of the system is formed in the postflexion larvae. However, the lateral-line canal of the body starts to develop in young juveniles from head to rear; the lateral-line canal extends to the vertical through the terminus of the dorsal-fin base in 60-day old specimens. Species of most genera of Pseudopimelodidae also present a well-developed lateral line that extends on the body from head to caudal-fin base. However, some species of the family have an incomplete lateral line wherein the lateral-line canal does not reach the caudal-fin base. This is the case in Microglanis and Batrochoglanis, wherein the lateral-line canal extends from the head to below the origin of the dorsal-fin base [9], or to the terminus of the adipose-fin base [33], respectively. The presence of an incomplete lateral line on the body is most likely due to a paedomorphic event, and is a putative synapomorphy for a clade composed of Batrochoglanis and Microglanis, corroborating previous hypotheses [9,11].
The skin of fully developed specimens of Lophiosilurus possesses many finger-like papillae which begin to develop as wart-like appendages in the postflexion larvae. These papillae are formed by an accumulation of keratin, and are distinct from the tubercles present in other catfishes [34,35,36,37] by lacking unculiferous cells [38]. The finger-like papillae probably function as camouflage for specimens of Lophiosilurus as they bury in the sand. Similar papillae are absent in most species of the family. Nevertheless, Cephalosilurus apurensis also has some papillae on the skin, indicating that it might be possibly closely related to Lophiosilurus, corroborating all three hypothesis of evolution of Pseudopimelodidae [9,11,10].
Species of most genera of Pseudopimelodidae, including Lophiosilurus, have an axillary gland that produces mucus and is located immediately beneath the anterior portion of the posterior cleithral process. In Lophiosilurus, this gland starts to develop in flexion stage larvae as a white tissue cluster immediately above the primary pectoral fin. However, in Batrochoglanis and Microglanis, the axillary gland is absent in adults, a synapomorphy for a clade uniting those genera [9,11], most likely due to a paedomorphic event.
The coloration of adult specimens of Lophiosilurus is greatly marked by the presence of widespread dark dendritic chromatophores, forming an overall dusky ground color. These dark chromatophores, although already present in yolk sac larvae, are only abundant in postflexion larvae, juveniles and adults. According to Nakatani et al. [39], most fishes have pelagic larvae that are usually scarcely pigmented, but undergo changes in pigmentation during the juvenile period when the fishes begin to explore different environments and need camouflage. However, as larvae of Lophiosilurus are benthic and guarded by the male in excavated nests [40,12], the change of coloration that takes place in the postflexion stage can be possibly coincident with the onset of parental care. The color pattern of most species of Pseudopimelodidae is formed by dark transverse bars on the body that is somewhat triangular, wide dorsally and tapered ventrally, and dark irregular blotches on the sides of head and body. The only exceptions are Lophiosilurus, as described above, and Cephalosilurus fowleri. Even though fully developed specimens of the latter have the coloration of Lophiosilurus, juveniles of C. fowleri have the color pattern typical of most species of Pseudopimelodidae. Therefore, the absence of dark bands and blotches in C. fowleri and L. alexandri can be interpreted as independently acquired, both conditions being autapomorphic.
The analysis presented herein shows that the study of the development of Lophiosilurus alexandri helps to determine the limits and polarity of the character states related to the evolution of the Pseudopimelodidae, especially for sister taxa relationships, such as those of Batrachoglanis and Microglanis, Lophiosilurus and Cephalosilurus, and Cruciglanis and Pseudopimelodus, conditions observed in larval and juvenile stages of L. alexandri illuminate how character states are connected, and their homologies across taxa in the family. Noteworthy to say that some of the features thought to be completely modified in adults of Lophiosilurus alexandri (autapomorphic), and therefore uninformative for the evolution of other species of Pseudopimelodidae, were in fact part of a complex character-state series that is traceable through ontogeny.