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Effects of Precaudal Elongation on Visceral Topography in a Basal Clade of Ray-Finned Fishes
Article first published online: 18 NOV 2011
Copyright © 2011 Wiley-Liss, Inc.
The Anatomical Record
Volume 295, Issue 2, pages 289–297, February 2012
How to Cite
Ward, A. B. and Kley, N. J. (2012), Effects of Precaudal Elongation on Visceral Topography in a Basal Clade of Ray-Finned Fishes. Anat Rec, 295: 289–297. doi: 10.1002/ar.21491
- Issue published online: 11 JAN 2012
- Article first published online: 18 NOV 2011
- Manuscript Accepted: 31 AUG 2011
- Manuscript Received: 20 FEB 2011
- National Science Foundation. Grant Numbers: IOS-0308746, IOS-0749750
- Department of Biology
- Adelphi University; Department of Anatomical Sciences
- Stony Brook University
- body elongation;
- gastrointestinal tract;
- anteroposterior patterning;
Elongate body forms have evolved numerous times independently within Vertebrata. Such body forms have evolved in large part via changes to the vertebral column, either through addition or lengthening of vertebrae. Previous studies have shown that body elongation in fishes has evolved most frequently through the addition of caudal vertebrae. In contrast, however, body elongation in Polypteriformes, a basal clade of ray-finned fishes (Actinopterygii), has evolved through the addition of precaudal vertebrae; one genus, Erpetoichthys, has approximately twice as many precaudal vertebrae as do members of its sister genus, Polypterus. Thus, polypteriform fishes provide an excellent opportunity to study the effects of precaudal elongation on the gross morphology and organization of visceral organs contained within the body cavity. In this study, we document the anteroposterior positions of most major visceral organs in representative species of both genera (E. calabaricus and P. palmas), relative to both vertebral number and percent pre-anal length. We found that, whereas the positions of the anterior and posterior borders of the visceral organs relative to percent pre-anal length were generally similar between the two species, most visceral organs were positioned further posteriorly in E. calabaricus than in P. palmas with respect to vertebral number. Based on previous determinations of the molecular control of anteroposterior patterning of the visceral organs, we discuss which possible changes in gene expression may have led to the anatomical modifications seen in the visceral morphology of Erpetoichthys. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.
Elongate body forms have evolved numerous times independently within Vertebrata (e.g., Wake, 1966; Richardson et al., 1998; Ward and Brainerd, 2007). Axial elongation occurs through a lengthening of the vertebral column, either by adding or lengthening vertebrae (Wake, 1966; Ward and Brainerd, 2007). Increases in vertebral number have evolved in nearly all major clades of vertebrates, and often such increases are restricted to specific regions along the vertebral column (Wake, 1966; Asano, 1977; Polly et al., 2001). That is, vertebrates may become elongate by adding vertebrae to one region of the vertebral column without changes to the other regions. Previous work has shown that body elongation in ray-finned fishes (Actinopterygii) is achieved most frequently through evolutionary increases in the number of caudal vertebrae (Ward and Brainerd, 2007). However, one striking exception to this generalization occurs within the basal actinopterygian family Polypteridae. Although both extant genera contained within this clade have similar numbers of caudal vertebrae, Erpetoichthys (reedfish) may have as many as twice the number of precaudal vertebrae as do members of its sister genus, Polypterus (bichirs) (Ward and Brainerd, 2007; Suzuki et al., 2010). This renders Polypteridae as an excellent model system in which to investigate the effects of precaudal elongation on the morphology of the visceral organs.
Polypteridae includes at least 16 extant species of African freshwater fishes (Nelson, 2006). All but one of these are included in the genus Polypterus; Erpetoichthys is monotypic, containing only the single species E. calabaricus. Although most species of Polypterus are relatively elongate in comparison with many other actinopterygian fishes, Erpetoichthys is significantly more elongate, exhibiting an almost snake-like body form (Fig. 1; see also Ward and Brainerd, 2007). Due in large part to their phylogenetic position as the most basal extant members of Actinopterygii (Inoue et al., 2003; Nelson, 2006), polypterids have received considerable attention from ichthyologists, and more widely, among vertebrate biologists in general (e.g., Britz and Bartsch, 1998; Chiu et al., 2004; Gemballa and Roeder, 2004; Hoegg et al., 2004; Claeson et al., 2007; Suzuki et al., 2010). However, few studies have sought to investigate body elongation in this clade (Ward and Brainerd, 2007; Suzuki et al., 2010), whereas many such studies have been conducted for numerous clades of tetrapods. Previous studies of elongate tetrapods, such as caecilians (Gymnophiona), amphisbaenians (Amphisbaenia), and snakes (Serpentes) have demonstrated that body elongation generally evolves concomitantly with radical modifications in the organization of internal organs (e.g., Bellairs, 1969; Duellman and Trueb, 1994). Thus, in this study, we examine the effects of extreme body elongation in Erpetoichthys on the morphology of its visceral organs.
The few previous studies of the visceral anatomy of polypterids have reported that the overall morphology of the gastrointestinal tract is relatively simple (Purser, 1929; Abdel-Aziz, 1957; Abdel Magid, 1975; Fig. 2). The esophagus opens into the stomach, which is U-shaped and bears a large gastric caecum (Purser, 1929; Abdel Magid, 1975). The intestine bends away from the stomach at the pylorus, after which it extends straight posteriorly. There is no looping of the gut in either Polypterus or Erpetoichthys (Purser, 1929; Abdel-Aziz, 1957). The liver of Erpetoichthys has been described as being long and thin, similar to the overall body form of that genus (Purser, 1929), whereas the liver of Polypterus is broad anteriorly and then narrows posteriorly (Abdel-Aziz, 1957). The common bile duct joins the intestine just posterior to the pyloric valve (Purser, 1929). The pancreas is closely associated with the ventral surface of the liver, as the two are contained within the same peritoneal sheath (Purser, 1929; Abdel-Aziz, 1957). All polypterids have two lungs: a short left lung and a long right lung (Goodrich, 1933). Despite this existing body of knowledge, however, there have been no comparative studies that have quantitatively documented the visceral topography of Erpetoichthys versus Polypterus, and thus the effects of body elongation on the organization of internal organs in this clade (and other fishes) remains poorly understood.
MATERIALS AND METHODS
Ten adult Erpetoichthys calabaricus (Smith, 1865) and 14 adult Polypterus palmas (Ayres, 1950), all from the personal research collection of ABW, were used for this study. All specimens were previously frozen and then thawed prior to being measured and dissected. Measurements of body length (pre-anal length (PAL), standard length (SL), and total length (TL); Table 1) were made to the nearest millimeter using a standard ruler, whereas other external measurements (Table 1) were made to the nearest 0.1 mm using digital calipers. Body mass was recorded to the nearest 0.1 g using a digital balance.
|Measurement/count||Erpetoichthys calabaricus (N = 10)||Polypterus palmas (N = 14)|
|Mass (g)||17.0 ± 1.8||39.9 ± 4.3|
|Maximum head width (mm)||9.6 ± 0.2||20.0 ± 0.8|
|Pre-anal length (mm)||231 ± 7.6||141 ± 5.1|
|Standard length (mm)||245 ± 8.0||166 ± 6.2|
|Total length (mm)||253 ± 8.4||198 ± 7.4|
|Body circumferencea (mm)||33.7 ± 1.3||65.4 ± 1.3|
|Body widtha (mm)||9.9 ± 0.3||18.8 ± 0.9|
|Body deptha (mm)||9.4 ± 0.4||18.9 ± 0.7|
|No. of precaudal vertebrae||103 ± 0.7||49 ± 0.8|
Fish were cut along the ventral midline from the anus to the gular plates. The cut edges of the body wall were then splayed laterally with dissection pins so that the internal organs could be observed without changing their positions. Positions of the following viscera were recorded: heart, right lung, stomach, liver, gallbladder, pancreas, kidney, and both right and left gonads (Fig. 2). Specifically, distances from the tip of the snout to the anterior and posterior borders of each organ were measured to the nearest millimeter using digital calipers. The location of the pylorus was also recorded (Fig. 2). After these measurements were taken, the liver was removed completely from the body cavity and its volume was measured to the nearest 0.5 mL via displacement of water using a 10-mL graduated cylinder.
After all measurements had been taken, the specimens were fixed in 10% neutral buffered formalin before being transferred into 70% ethanol for permanent storage. Each specimen was X-rayed using a Faxitron 43855 cabinet X-ray machine. The X-ray negatives were then digitally scanned for further analysis. The position of each organ was traced onto the corresponding X-ray image digitally using the measurement tools available in ImageJ 1.34 (available at http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, Bethesda, USA). The closest vertebra to each such position was recorded for every anatomical landmark.
Two sets of comparisons were made between Erpetoichthys calabaricus and Polypterus palmas to examine the effects of body elongation on visceral topography. We recorded the positions of the anterior and posterior borders of major visceral organs first with respect to vertebral number, and second with respect to percent pre-anal length. Data were analyzed using the statistical software package JMP (v. 5.1, SAS Institute, Cary, NC). We tested all data for both normality (using the Shapiro-Wilk test) and homoscedasticity (using the Levene test). If the data were normal and homoscedastic, we used a parametric Student's t-test to compare species means. If the data were non-normal and/or heteroscedastic, a nonparametric Wilcoxon/Kruskal-Wallis test was used. Means were considered to be significantly different if the P-values derived from these tests were less than 0.05. Liver volume was regressed against body mass using ordinary least squares (OLS) regression, and analysis of covariance (ANCOVA) was used to determine whether species differed significantly in the scaling relationship of liver volume to body mass.
The specimens of Erpetoichthys calabaricus that we examined in this study (mean SL = 245 mm) were significantly longer than those of Polypterus palmas (mean SL = 166 mm). In contrast, however, body depth, width, and circumference at 50% pre-anal length were significantly greater in P. palmas than in E. calabaricus; these measurements in P. palmas were approximately twice those in E. calabaricus (Table 1, Fig. 1).
Visceral Topography Relative to Vertebral Number
The mean number of precaudal vertebrae in our sample of Erpetoichthys calabaricus was 103 (±0.7 S.E.), whereas that of Polypterus palmas was 49 (±0.8 S.E.) (Table 2). The vertebral position of the anterior border of each visceral organ examined was found to be significantly different between E. calabaricus and P. palmas (Table 2, Fig. 3). Similarly, the vertebral position of the posterior border of each organ was also found to be significantly different between the two species. The positions of organs in E. calabaricus were more posterior than those in P. palmas; that is, the anterior and posterior borders of all major visceral organs in E. calabaricus were found to be positioned subjacent to vertebrae of higher numbers (as counted from anterior to posterior) than those in P. palmas.
|Anatomical landmark||Erpetoichthys calabaricus||Polypterus palmas||Significance level|
|Lung (anterior)||1.5 ± 0.3||0 ± 0||P < 0.001|
|Lung (posterior)||102.8 ± 0.7||48.7 ± 0.8||P < 0.001|
|Lung (span)||101.3 ± 0.8||48.7 ± 0.8||P < 0.001|
|Heart (anterior)||1.5 ± 0.3||0 ± 0||P < 0.001|
|Heart (posterior)||3.0 ± 0.4||1.9 ± 0.3||P < 0.05|
|Heart (span)||1.5 ± 0.3||1.9 ± 0.3||P > 0.05|
|Stomach (anterior)||41.6 ± 0.7||14.0 ± 0.5||P < 0.001|
|Stomach (posterior)||58.8 ± 1.1||31.2 ± 0.9||P < 0.001|
|Stomach (span)||17.2 ± 0.8||17.2 ± 1.0||P > 0.05|
|Pylorus||37.1 ± 0.8||12.6 ± 0.5||P < 0.001|
|Liver (anterior)||17.0 ± 1.5||4.5 ± 0.3||P < 0.001|
|Liver (posterior)||93.3 ± 2.1||38.1 ± 0.8||P < 0.001|
|Liver (span)||76.3 ± 2.9||33.6 ± 0.7||P < 0.001|
|Pancreas (anterior)||44.6 ± 1.1||15.6 ± 0.6||P < 0.001|
|Pancreas (posterior)||85.7 ± 2.6||36.1 ± 0.6||P < 0.001|
|Pancreas (span)||41.1 ± 2.9||20.4 ± 0.4||P < 0.001|
|Gallbladder (anterior)||35.9 ± 0.8||9.2 ± 0.5||P < 0.001|
|Gallbladder (posterior)||38.1 ± 0.9||12.6 ± 0.5||P < 0.001|
|Gallbladder (span)||2.2 ± 0.2||3.4 ± 0.3||P < 0.01|
|Kidney (anterior)||11.7 ± 1.3||3.9 ± 0.6||P <0.001|
|Kidney (posterior)||102.8 ± 0.7||48.7 ± 0.8||P < 0.001|
|Kidney (span)||91.1 ± 1.5||44.8 ± 0.8||P < 0.001|
|Right ovary (anterior)||51.0 ± 3.4||13.3 ± 0.9||P < 0.01|
|Right ovary (posterior)||83.8 ± 1.7||32.5 ± 1.3||P < 0.01|
|Right ovary (span)||32.8 ± 4.1||19.3 ± 0.9||P > 0.05|
|Left ovary (anterior)||52.6 ± 2.8||18.7 ± 1.8||P < 0.01|
|Left ovary (posterior)||87.6 ± 2.5||32.0 ± 1.5||P < 0.01|
|Left ovary (span)||35.0 ± 1.6||13.3 ± 0.9||P < 0.01|
|Right testis (anterior)||26.0 ± 0.9||15.3 ± 1.2||P < 0.05|
|Right testis (posterior)||27.4 ± 0.9||17.5 ± 0.6||P < 0.05|
|Right testis (span)||1.4 ± 0.2||2.3 ± 0.9||P > 0.05|
|Left testis (anterior)||28.2 ± 1.2||16.0 ± 0.8||P < 0.01|
|Left testis (posterior)||30.2 ± 1.2||20.2 ± 1.2||P < 0.01|
|Left testis (span)||2.0 ± 0.0||4.2 ± 1.0||P < 0.05|
We also recorded the vertebral span of each organ (Table 2, Fig. 3). We found that the lung, liver, pancreas, kidney, left ovary, and left testis span more vertebrae in Erpetoichthys calabaricus than in Polypterus palmas. More specifically, the vertebral spans of these organs in E. calabaricus were approximately twice those seen in P. palmas. However, this was not found to be the case for the heart, stomach, gallbladder, right ovary, or right testis. The heart, stomach, right ovary, and right testis did not differ significantly between the two species in the number of vertebrae spanned, and the gallbladder actually spanned fewer vertebrae in E. calabaricus than in P. palmas.
Visceral Topography Relative to Percent Pre-anal Length
In addition to determining the positions of visceral organs relative to vertebral number, we also recorded the positions of these organs as percentages of pre-anal length (PAL) (Table 3, Fig. 4). We found that the anterior borders of the lung, heart, stomach, pylorus, liver, pancreas, kidney, and testes differed significantly in percent PAL position between Erpetoichthys calabaricus and Polypterus palmas. In contrast, however, we found no significant differences in the positions of the anterior borders of the gallbladder or ovaries. Significant differences were also found between E. calabaricus and P. palmas in the positions of the posterior borders of the heart, stomach, liver, gallbladder, and testes, whereas the lung, pancreas, kidney, and ovaries exhibited no such differences. The percent PAL spans of the lung, liver, pancreas, kidney, and left ovary were significantly greater in E. calabaricus than in P. palmas, whereas the reverse was true for the heart, stomach, gallbladder, and testes. The only organ that did not differ significantly in percent PAL between the two species was the right ovary.
|Anatomical landmark||Erpetoichthys calabaricus||Polypterus palmas||Significance level|
|Lung (anterior)||6.62 ± 0.28||16.58 ± 0.32||P < 0.001|
|Lung (posterior)||100 ± 0||100 ± 0||P = 1.0|
|Lung (span)||93.38 ± 0.28||83.42 ± 0.32||P < 0.001|
|Heart (anterior)||6.62 ± 0.28||16.58 ± 0.32||P < 0.001|
|Heart (posterior)||8.12 ± 0.30||24.72 ± 0.46||P < 0.001|
|Heart (span)||1.50 ± 0.17||8.13 ± 0.42||P < 0.001|
|Stomach (anterior)||41.59 ± 0.45||43.87 ± 0.50||P < 0.01|
|Stomach (posterior)||57.69 ± 1.08||71.91 ± 1.53||P < 0.001|
|Stomach (span)||16.10 ± 0.82||28.04 ± 1.69||P < 0.001|
|Pylorus||37.43 ± 0.57||40.99 ± 0.41||P < 0.001|
|Liver (anterior)||19.76 ± 1.32||29.05 ± 0.32||P < 0.001|
|Liver (posterior)||91.55 ± 1.35||83.71 ± 0.85||P < 0.001|
|Liver (span)||71.79 ± 2.22||54.66 ± 0.88||P < 0.001|
|Pancreas (anterior)||44.38 ± 0.72||46.74 ± 0.53||P < 0.01|
|Pancreas (posterior)||84.49 ± 2.54||80.19 ± 0.76||P > 0.05|
|Pancreas (span)||40.11 ± 2.82||33.44 ± 0.64||P < 0.05|
|Gallbladder (anterior)||36.25 ± 0.62||36.91 ± 0.44||P > 0.05|
|Gallbladder (posterior)||38.10 ± 0.94||42.07 ± 0.49||P < 0.001|
|Gallbladder (span)||1.97 ± 0.23||5.17 ± 0.33||P < 0.001|
|Kidney (anterior)||15.11 ± 1.34||28.17 ± 0.64||P < 0.001|
|Kidney (posterior)||100 ± 0||100 ± 0||P = 1.0|
|Kidney (span)||84.89 ± 1.34||71.83 ± 0.64||P < 0.001|
|Right ovary (anterior)||47.51 ± 1.17||44.17 ± 1.74||P > 0.05|
|Right ovary (posterior)||82.66 ± 1.32||76.28 ± 4.53||P > 0.05|
|Right ovary (span)||35.15 ± 2.07||32.12 ± 3.17||P > 0.05|
|Left ovary (anterior)||52.17 ± 2.37||52.87 ± 5.19||P > 0.05|
|Left ovary (posterior)||86.57 ± 1.80||77.08 ± 5.39||P > 0.05|
|Left ovary (span)||34.40 ± 1.81||24.21 ± 2.28||P < 0.05|
|Right testis (anterior)||27.22 ± 0.58||46.42 ± 1.00||P < 0.001|
|Right testis (posterior)||28.63 ± 0.66||50.78 ± 1.29||P < 0.001|
|Right testis (span)||1.41 ± 0.16||4.36 ± 1.09||P < 0.05|
|Left testis (anterior)||29.09 ± 0.74||47.46 ± 1.16||P < 0.001|
|Left testis (posterior)||30.84 ± 0.83||53.81 ± 0.78||P < 0.001|
|Left testis (span)||1.76 ± 0.13||6.35 ± 1.68||P < 0.01|
To assess whether the relative volume of a given organ differed between Erpetoichthys calabaricus and Polypterus palmas, the livers were removed from the bodies of all specimens prior to fixation and their volumes were measured. Regressions of liver volume on body mass were significant for both species (Fig. 5). However, the slopes were not significantly different from one another (P > 0.05), nor were the y-intercepts (P > 0.05), indicating that relative liver volume does not differ significantly between the two species (in contrast to the percent pre-anal length of the liver, which was found to differ significantly between these two species [see above]).
Two extant genera are contained within Polypteridae: Erpetoichthys is a highly elongate eel- or snake-like form, whereas the 15 species of Polypterus are more stout-bodied (Fig. 1). Body elongation in Erpetoichthys results predominantly from an approximately two-fold increase in the number of precaudal vertebrae relative to that seen in Polypterus species (Ward and Brainerd, 2007; this study). In this study, we examined the effects of this doubling of the number of precaudal vertebrae on the anteroposterior patterning of the major visceral organs.
Visceral Anatomy in Fishes
Visceral anatomy has been described for many different species of actinopterygian fishes. There are a number of modifications of the gastrointestinal tract in certain fishes, in particular to the stomach and intestine, for both digestion and respiration. For instance, osteoglossomorph fishes have an increased number of flexures of the intestine, as has the sandsmelt, Atherinomorus lacunosus (Al-Husaini, 1947; Nelson, 1972). Similarly, a variety of catfishes exhibit a number of modifications of the viscera associated with respiration; for example, the stomach is greatly expanded in certain loricariid catfishes, such as Pterygoplichthysanisitsi and Hypostomus panamensis (Carter and Beadle, 1931; Armbuster, 1998). Pyloric caeca are common in fishes, and differ in number and size (Aristotle, 1955; Weinreb and Bilstad, 1955; Buddington and Diamond, 1986). Given the wide diversity in the form of the viscera in fishes, there is a wealth of possible species for examining the molecular control of patterning of the viscera.
Visceral Topography and Body Elongation
Previous studies of anteroposterior patterning have focused primarily on modifications to mesodermal derivatives, in particular the vertebral column (e.g., Polly et al., 2001; Narita and Kuratani, 2005; Ward and Brainerd, 2007). Such studies have led to a number of evolutionary hypotheses concerning the developmental control of the various regions of the vertebral column. Despite numerous developmental studies on the molecular control of regionality within the endoderm (e.g., Grapin-Boton, 2005; Dessimoz et al., 2006; Kimura et al., 2007), there are few morphological studies that have investigated basic patterning of major visceral organs (e.g., Brongersma, 1951; Bergmann, 1953, 1955; Starck, 1999). In this study, we consider the effects of an evolutionary increase in the number of precaudal body segments on the topography of the viscera contained within the body cavity.
Our study addressed two aspects of visceral topography: organ position and how many vertebrae each organ spans. First, we analyzed the position of each organ within the body cavity and tested whether there are differences between Erpetoichthys and Polypterus to determine whether precaudal elongation affected the anteroposterior patterning of the visceral organs. Secondly, we tested the hypothesis that doubling the number of precaudal vertebrae in Erpetoichthys would result in organs that span twice as many vertebrae in Erpetoichthys than in Polypterus.
The anterior and posterior positions of each organ were measured and compared between Erpetoichthys calabaricus and Polypterus palmas. (Tables 2 and 3; Figs. 3 and 4). The anterior and posterior vertebral positions of every visceral organ studied were found to differ significantly between these two species. All structures were located more posteriorly in E. calabaricus than in P. palmas relative to vertebral position (Table 2). This indicates that there is likely an early change in patterning of the visceral organs.
Despite the vertebral positions of visceral organs differing dramatically between Erpetoichthys and Polypterus, we found that, in general, organs were located in similar positions in Erpetoichthys and Polypterus when standardized by percent pre-anal length (Table 3, Fig. 4). Although the anterior and posterior positions did differ significantly, there was significant overlap in the relative locations of most structures in the two species. The notable exceptions to this generalization are the heart and testes; both of these organs are located more anteriorly in Erpetoichthys than in Polypterus. The similarity in percent pre-anal location indicates that, despite the significant difference in number of segments, the overall arrangement of the visceral organs is similar between the two genera. This suggests that segmental identity is important for anteroposterior patterning of the visceral organs, as has been shown previously for vertebral development (Burke et al., 1995; Grapin-Boton, 2005).
Vertebral Span and Organ Size
We found that when the vertebral span of organs was greater in Erpetoichthys than in Polypterus, the number of vertebrae spanned was approximately twice as many in Erpetoichthys than in Polypterus. This was the case for the lung, liver, pancreas, and kidney (Fig. 3). The left ovary spanned over 2.5 times as many vertebrae in Erpetoichthys than in Polypterus. The other organs examined either spanned approximately the same number of vertebrae in each genus, or the organ in Polypterus spanned more vertebrae than in Erpetoichthys, as with the gallbladder and left testis (Fig. 3, Table 2). The finding that the lung and kidney span twice as many vertebrae in Erpetoichthys than in Polypterus is perhaps not surprising because these organs span the length of the body cavity; both organs have their posterior limit at the cloaca.
Among the specimens examined in this study, the left ovary in Erpetoichthys spanned more than twice as many vertebrae as in Polypterus. However, this might have been due to a lack of sexual maturity in the specimens of Polypterus that we examined. All of the female Erpeotichthys that we examined contained a large number of eggs in their ovaries. However, none of the female Polypterus we examined had eggs in their ovaries. It is possible that if female specimens of Polypterus had been carrying eggs, the results would have been more similar to those found for the testes, which spanned more vertebrae in Polypterus than in Erpetoichthys. The left testis and gallbladder were the only structures that spanned fewer vertebrae in Erpetoichthys than in Polypterus. Similarly, the percent pre-anal lengths of these organs were also less in Erpetoichthys than in Polypterus.
The heart, stomach, and right gonads span approximately the same number of vertebrae in Erpetoichthys calabaricus and Polypterus palmas. Nevertheless, the relative sizes these organs, as measured by percent pre-anal length with the exception of the right ovary, is greater in Polypterus than in Erpetoichthys. Having a wider head, Polypterus is more likely to eat larger prey than an Erpetoichthys of similar standard length, which may be the reason for the former having a relatively larger stomach. Species of Polypterus tend to be piscivorous, while also feeding on insects and crustaceans, whereas Erpetoichthys tends to feed on small worms and insects (e.g., Reed et al., 1967; Mills and Vevers, 1989). It is also possible that stomach size could be affected by changes in nutritional status or diet. For example, gut length has been shown to increase or decrease depending on diet in Eurasian perch (Perca fluviatilis; Olsson et al., 2007).
We were able to measure the volume of only one visceral organ, the liver. When regressed against body mass, liver volume was found to scale similarly in Erpetoichthys and Polypterus. The liver of Polypterus is wide at the anterior end and narrows posteriorly, whereas the liver in Erpetoichthys is narrow along its entire its span. This finding is notable because of the extreme differences in span of the liver. The liver spans twice as many vertebrae and a greater proportion of the abdominal cavity in Erpetoichthys than in Polypterus (Tables 2 and 3). Despite these differences, the overall volume is similar for similarly sized individuals. This implies that, while visceral shape and size may differ between these species, there is a conservation of organ volume. Roux (1922) hypothesized that the liver will fill the available space in the visceral cavity. It is therefore surprising, however, that another highly elongate species, Monopterus albus (Synbranchidae), has a liver that extends only to the pylorus and does not run the length of the visceral cavity (Liem, 1967).
Developmental Patterning of Visceral Organs
This study provides a basic methodology for analyzing changes in anteroposterior patterning among visceral organs. Unlike the axial skeleton, less study has focused on the segmental identity of anatomical boundaries within the body cavity. Studies of the anteroposterior patterning of the digestive tract have demonstrated that, as in the axial skeleton, there are distinct boundaries that are established during early development. Fate-mapping studies have shown that the anteroposterior pattern of the endoderm is present as early as mid-gastrulation in zebrafish (Warga and Nüsslein-Volhard, 1999; Ward et al., 2007).
The gastrointestinal tract is patterned into three primary anteroposterior regions; the foregut is defined by Hhex/Sox2, the midgut by Pdx1, and the hindgut by Cdx (for a recent review, see Zorn and Wells, 2009). Several factors can affect the boundary between Pdx1 and Cdx, including retinoic acid and FGF (Dessimoz et al., 2006; Kinkel et al., 2008, 2009; Bayha et al., 2009). Addition of exogenous retinoic acid causes posteriorization of the pancreas and liver, resulting in cells that are found in more anterior locations than during normal development (Stafford and Prince, 2002). In Erpetoichthys calabaricus, in which the same organ is generally located at more posterior vertebral locations than in Polypterus palmas, it is possible that a similar mechanism during early development affects the positioning of at least some of the visceral organs.
Kinkel et al. (2008) demonstrated that misexpression of cdx4 resulted in anteriorization of the developing gut tube. The anterior boundaries of most visceral organs are located at more anterior segment numbers in Polypterus than in Erpetoichthys, even though the relative positions (i.e., relative to percent pre-anal length) of the organs are similar (Figs. 3 and 4). It is possible that in the lineage that led to E. calabaricus, there was a modification in expression of Cdx, allowing for anteriorization of the body cavity in this species. If the anterior extent of the Cdx expression boundary is located more posteriorly, this would lead to visceral structures being located at higher vertebral numbers. Cdx signaling is also related to the number of somites. Decrease in cdx1a and cdx4 results in truncation of the body in zebrafish (Shimizu et al., 2005). Thus, there is likely a link between the changes related to elongation of the axial skeleton and associated changes among the visceral organs within the body cavity.
However, it is also possible that the differences in segmental identity of the visceral organs are due, in part, to ontogenetic changes. Previous studies of snakes have documented ontogenetic changes in the positions of visceral organs (e.g., Bergman 1953, 1955, 1958). For example, in adults of Xenopeltis unicolor and Cylindrophis ruffus, there is anteriorization of the major visceral organs (pancreas, spleen, gallbladder, gonads, and kidney) in comparison to juveniles (Bergman 1953, 1955). Similarly, in Acrochordus javanicus, there is ontogenetic anteriorization of the liver, spleen, gallbladder, and gonads (although this anteriorization is based on the relative position of the organs; Bergman, 1958). The extreme anteriorization of major visceral organs in Erpetoichthys relative to Polypterus may be related to postembryonic growth as in many snake species.
In this study, we considered the effects of an evolutionary doubling of the number of precaudal vertebrae on patterning of the major internal organs in Erpetoichthys. We found that increasing the number of precaudal vertebrae has a significant effect on the segmental (i.e., vertebral) positions of the viscera, but only slight effects on the percent pre-anal positions of the viscera within the body cavity (Figs. 3 and 4). This study provides an initial baseline for understanding the relationship between precaudal elongation and visceral topography within the body cavity of vertebrates.
The authors thank M. Bell for the use of the Faxitron cabinet X-ray machine, and also thank two reviewers whose comments improved this manuscript.
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