Ontogeny of Functional Design in Tiger Salamanders (Ambystoma Tigrinum

JOURNAL OF MORPHOLOGY 197249-268 (1988)

Ontogeny of Functional Design in Tiger Salamanders (Ambystoma tigrinurn):Are Motor Patterns Conserved During Major Morphological Transformations?

GEORGE V. LAUDER AND H. BRADLEY SHAFFER School of Biological Sciences, University of California, Irvine, California 9271 7 ABSTRACT The process of metamorphosis in tiger salamanders, Ambys- toma tigrinum, is used to investigate motor pattern conservatism in vertebrates. Specifically,we examined cranial muscle activity to determine if changes in the motor pattern are correlated with the morphological or environmental changes that occur at metamorphosis. Twenty-three variables were measured from electromyographic recordings from six cranial muscles in 13 tiger salamanders. These variables described the configuration of the motor pattern: the peak amplitude of activity, duration, relative onset, and time to peak amplitude were measured for each of the six muscles. Univariate and multivariate statistical analyses showed that there was no change in the mean motor pattern associated with the morphological transformation at metamorphosis: larval and metamorphosed individuals feeding in the water have very similar motor patterns. This was true despite significant morphological changes in the design of the feeding mechanism at metamorphosis and despite a significant decrease in aquatic feeding performance following metamorphosis. There was a change in the mean motor pattern to jaw muscles when metamorphosed individuals fed in water and on land: metamorphosed terrestrial feedings tend to have longer bursts of muscle activity then do aquatic feedings. The environmental changes in the motor pattern cannot be attributed to effects of differing fluid density or viscosity between water and air and are instead related to the shift to feeding by tongue projection on land. The decrease in aquatic feeding performance that occurs after metamorphosis is not correlated with changes in the motor pattern. Instead, the results suggest that changes in behavioral performance during ontogeny are associated with the transformation of hydrodynamic design of the feeding mechanism from uni- to bidirectional, and that motor patterns driving complex rapid behaviors may be conserved when behavior is altered by changes in peripheral morphology.

How does the behavior of animals change will result in different behaviors. By behav- in ontogeny and phylogeny? One approach ior we mean the actions or movements of to this general problem is to consider the animals (Lauder, '86, '88), and such actions levels of biological design that might be re- are often quantified by measuring animal sponsible for changes in behavior. performance (e.g., Arnold, '83; Emerson and Diiyerences in behavior between two spe- Diehl, '80; Reilly and Lauder, '88a). Alter- cies, for example, might be based only on natively, differences between two species in differences in the structure and topography the timing and sequence of muscle activity of their musculoskeletal systems. All other things being equal, differences between species in muscle masses, origins and inser- H. Bradley ShafFer is now at Dept. of Zoology, Univ. of Cali- tions, physiological properties, and lever arms fornia, Davis, Davis, CA 95616.

($1 1988 ALAN R. LISS, 1NC 250 G.V. LAUDER AND H.B. SHAFFER

Ambvstoma tierinum ments to test the hypothesis that the correlated changes between behavior and mo- Larval Metamorphosed Metamorphosed tor patterns are in fact causally related. Any aquatic terrestrial observed changes in organismal morphology or physiology must experimentally be shown to account for changes observed in behavior. Previous research on lower vertebrate mo- Environment Morphology constant constant tor patterns (Bemis and Lauder, '86; Lauder, '80, '83; Lauder and Shaffer, '85) has ex- Behavior simjlar: Behavior different differences in amined one of the three steps, has not used efficiency this overall research strategy, and instead has focused on a comparative, phylogenetic Fig. 1. Schematic diagram of the experimental plan analysis of motor patterns in relation to used in this study. Ambystoma tigrinum will feed under three conditions: as larvae in the water, as metamor- modifications in feeding behavior. This work phosed animals in the water, and as metamorphosed has indicated that motor patterns may be animals on land. By comparing the motor pattern be- conservative across a wide phylogenetic tween larval and metamorphosed animals feeding in the range. For example, Lauder ('80; also see water (test l),we test only for the effect of morphological changes occurring at metamorphosis on the motor pat- '85) has emphasized that the feeding system tern. As shown by Shaffer and Lauder ('88), the aquatic of ray-finned fishes as phylogenetically di- feeding kinematics used by these two stages are similar, verse as bichirs (Polypterus),gars (Lepisos- hut they differ in efficiency (Lauder and Shaffer, '86). teus), and bowfins (Amia) shows consider- By comparing the motor pattern used by metamorphosed individuals feeding in the water and on land able similarities in the motor patterns to the (Test 2), we test only for the effect of the environmental jaw muscles, despite major morphological change on the motor pattern. Feeding kinematics are changes in the structure of the muscles and different (Lauder and Shaffer, '86) between these two bones of the feeding mechanism. stages. The conclusions derived from these comparative studies of fish feeding systems (Lauder, '80, '85) have been reached by mak- (the motor pattern) could produce different ing comparisons within a single environ- behaviors even if the morphology of the mus- ment. Thus, the motor pattern to the jaw culoskeletal system is identical. Changes at muscles in fishes may be conservative be- the level of patterned outflow from the cen- cause the density and viscosity of water may tral nervous system to the peripheral mus- limit the range of possible functional solu- culature may in turn be the result of changes tions to the problem of prey capture. Simi- in central neuronal interconnections and larly, the lack of motor pattern and kine- morphology. Thus, two species may differ in matic diversity in terrestrial feeding systems behavior because of changes at any one or (Bramble and Wake, '85) may be due to the more of at least four levels: peripheral mor- constraints of feeding in air. phology, physiological properties of periph- The goal of this paper is to describe the eral structures, motor pattern, and central ontogeny of motor patterns in the tiger sal- neuronal connections and circuits. amander, Ambystoma tigrinum, and to in- The general research strategy that we have terpret the results in the framework of the used to pursue the issue of behavioral trans- three-part research strategy outlined above. formation involves three steps. The first step We have selected the tiger salamander for is the documentation of a change (or lack several reasons. First, A. tigrinum under- thereof) in behavior and animal perfor- goes a dramatic morphological reorganiza- mance. The second step is the examination tion in the skull and hyobranchium at met- of the various underlying levels mentioned amorphosis. Larval life in the water typically above for correlated changes. Thus, if' onto- lasts from 3 to 4 months, whereas adult ter- genetic stages A and B in an animal differ restrial life may extend for more than 10 in behavior, we propose to test for mean dif- years. This metamorphosis provides a major ferences in musculoskeletal morphology and change in morphology across which to ex- motor pattern between stages A and B. If amine motor patterns. By using ontogenetic any significant differences are found, then alterations in behavior, it is possible to avoid they can only be said to be correlated with many of the problems inherent in a com- the documented change in behavior. Third, parative analysis of different species. Sec- if correlated changes are found, then it is ond, the process of metamorphosis in uro- reasonable to conduct manipulative experi- deles, although representing a considerable ONTOGENY OF MOTOR PATTERNS 251

Fig. 2. Lateral views showing the external mor- tion in head depth, and the change in location of the phology of the head in Ambystoma tzgrinurn at three eyes. The gular membrane, which in larvae provides a times during ontogeny. A Larval. B: Mid-metamorphic. posterior exit of water from the buccal cavity, is sealed C.Postmetamorphic (adult). Note the reduction and loss to the body wall in adults. Scale bar = 2.0 cm. of the external gills during metamorphosis, the reduc- TABLE 1. Experimental design for longitudinal and cross-sectional electromyographic data on prey capture in Ambystoma tigrinum’ Individual Longitudinal Cross-sectional Metamorphic stage 4 7 5 9 12 13 8 11 17 6 10 14 23

Larval X X xx xx Metamorphosed x X xx X aquatic Metamorphosed x X xxxx terrestrial ‘Salamander identification No. is shown at the top of each column. An “x” indicates that data were obtained for that individual.

TABLE 2. Summary statistics for 23 electromyographic (EMG) variables measured from prey capture events (captures only) in Ambystoma tigrinum: Mean, sd (N)l Metamorphic stage EMG Metamorphosed Metamorphosed variable Larval aquatic terrestrial DMMAX .45, .13 (43) 55, .12 (16) .34, .15 (31) .37, .15 (22) .50, .11 (13) 53, .ll (21) DMTMAX 17. 16 (43) 24. 13 (16) 25, 19 (31) 10; 12 (22) 23; 15 (13) 43, 33 (20) DMDUR 87, 36 (43) 123, 55 (16) 81, 18 (31) 62, 24 (22) 102, 30 (13) 124, 38 (20) EPON - 1, 3 (43) 2, 10 (16) - 2, 8 (31) - 1, 2 (22) -7. 6 (13) 4. 5 (21) EPMAX .36. .07 (43) .40, .ll (16) .37,’ .08 (31) .37; .08 (22) .46, .04 (13) .35, .09 (21) EPTMAX 11, 9 (43) 36, 21 (16) 44, 29 (31) 17, 16 (22) 26, 22 (13) 41, 31 (21) EPDUR 68, 29 (43) 82, 25 (16) 102. 35 (31) 65, 26 (22) 91, 46 (13) 111, 39 (21) RCON 3, 3 (43) 9, 9 (16) - 1, 7 (31) 0. 3 (22) 2. 9 (13) 0, 6 (21) RCMAX .32, .18 (43) .37, .18 (16) .36, .15 (31) 55, .13 (22) .21, .15 (13) .31, .14 (21) RCTMAX 14, 10 (43) 26, 16 (16) 37, 46 (31) 11, 8 (22) 15, 5 (13) 26. 29 (21) RCDUR 48, 20 (43) 69. 15 (16) 112, 56 (31) 57. 18 (22) 56, 30 (13) 101, 32 (21) AMEON 0,3 (43) 14, 5 (5) 0, 4 (30) 4, 13 (22) 2, 8 (13) 1, 6 (21) AMEMAX .50, .16 (43) .41, .22 (5) .34, .18 (26) .27, .17 (22) .45, .16 (13) 50, .15 (21) AMETMAX 46, 26 (43) 117, 114 (5) 69. 47 (26) 54, 29 (22) 79, 57 (13) 173,-201ji1) AMEDUR 106, 36 (43) 165, 146 (5) 180, 150 (30) 87, 42 (22) 176, 253 (13) 338, 384 (21) AMION 1, 2 (43) 4, 16 (14) - 1, 5 (30) 5, 15 (20) 18, 25 (13) - 1, 6 (21) AMIMAX 53, .14 (43) .32, .17 (14) .24, .19 (26) .40, .21 (20) .38, .16 (13) 51. ,151 (21) AMITMAX 46. 21 (43) 85. 84 (14) 83,’47 (26) 41: 30 (20) 63; 38 (13) 161, 188 (21) AMIDUR 105, 43 (43) 139, 105 (14) 170, 69 (30) 77, 39 (20) 163, 181 (13) 434, 298 (21) BHiSARlON - 1, 2 (41) 13, 12 (15) 4. 4 (31) - 1, 4 (22) 9, 11 (13) 2, 4 (21) BHiSARlMAX .48, .13 (41) .30, .17 (15) .16, .09 (30) .46, .ll (22) .20, .14 (13) .43, .ll (21) BHiSARlTMAX 21, 21 (41) 44, 21 (15) 38, 20 (30) 9, 16 (22) 32, 18 (13) 46, 26 (21) BH/SARl DUR 126, 93 (41) 92, 38 (15) 89, 28 (31) 67, 27 (22) 62, 26 (13) 122, 61 (21) ‘Statistics for the cross-sectional data set are shown above the corresponding values for the longitudinal data set. Statistics are means for all individuals in each data set. All units are milliseconds except for the six variables that measure EMG amplitude (DMMAX, EPMAX, RCMAX, AMEMAX, AMIMAX, BHMAX), which are in millivolts. u Fig. 3. Dorsal view of the head in Ambystoma ti- C: Postmetamorphic (adult).Note the reduction in head grinurn to show the change in external shape at three width and the loss of external gills. Scale bar = 2.0 cm. times during ontogeny. A: Larval. B: Mid-metamorphic. modification, is not so great that it is difficult (Lumbricus)cut into 1- to 2-cm-long pieces. to homologize muscles across the transfor- During the experiments, the pieces of earth- mation. Thus, one can record from homolo- worm prey were presented to the animal on gous muscles throughout ontogeny. Third, the end of a pair of forceps in a similar man- although metamorphosis normally entails ner to that used in previous studies (Lauder an environmental transition from aquatic and Shaffer, '85, '86; Shaffer and Lauder, feeding to terrestrial prey acquisition, it is '88). Nine of the animals used for the elec- also possible to study the effect of metamor- tromyographic experiments were the same phosis on the motor pattern in the aquatic individuals used for a study of the meta- environment alone. Metamorphosed indi- morphosis of feeding kinematics (Shaffer and viduals will feed both in the water and on Lauder, '88) to allow direct individual com- land, allowing us to test for mean differences parisons of kinematics and motor patterns. in the motor pattern between aquatic and Animals of similar size were chosen to min- terrestrial feedings in the same individual imize overall size effects: morphological (Fig. 1).Fourth, the other analyses required measurements (in cm) of the 13 animals dur- by the research strategy outlined above have ing the experimental period were (mean, sd) been completed (Lauder and Reilly, '88; Lau- snout-vent length, 9.7,0.56; head width, 2.45, der and Shaffer, '86; Reilly and Lauder, '88a; 0.33. Shaffer and Lauder, '881, allowing us to pre- Gross morphological features of the cra- sent here a study of the motor patterns that nial musculoskeletal system were studied by completes the three-step analysis of the dissection of larval and metamorphosed Am- mechanisms underlying ontogenetic changes bystoma tigrinum from the same population of behavior in Ambystoma tigrinum. as the experimental animals. A Zeiss IVB dissecting microscope was used with a cam- MATERIALS AND METHODS era lucida to illustrate musculoskeletal Specimens and morphology anatomy. Several specimens at larval, mid- Electromyographic data were acquired metamorphic, and adult ontogenetic stages from 13 individual Ambystoma tigrinum col- were cleared and double stained for bone and lected in Lincoln Co., Colorado. All individ- cartilage following the procedures of Din- uals were collected as larvae, transported to gerkus and Uhler ('77). the laboratory, and then held individually Osteological and myological nomencla- at 1'7°C in 40-liter aquaria until metamor- ture follows that used by Druner ('021,Duell- phosis occurred naturally. After metamor- man and Trueb ('86), Edgeworth ('35), Fran- phosis, individuals were kept in either shal- cis ('34), Luther ('141, Piatt ('38, '39, '40), low water (about 4 cm deep) with rocks to Reilly and Lauder ('88b), and Wilder ('25). allow them access to the air, or in aquaria In addition, we consider (following Smith, with moist paper towels. During both the '20) that the metamorphosed subarcualis experimental and holding periods salaman- rectus one muscle (Fig. 7: SARl) is homol- ders were fed a diet of live earthworms ogous to the muscle of that name in the larva 254 G.V. LAUDER AND H.B. SHAFFER

(Fig. 6: SARl), and not to the muscle that motor pattern between the two environ- occupies a similar anatomical position in the ments. larva, the branchiohyoideus (Fig. 6: BH; Two statistical experimental designs were Lauder and Shaffer, '85; = ceratohyoideus used to implement the conceptual plan out- externus of Driiner, '02). Because of the sim- lined in Figure 1, and both univariate and ilarities in line of action and function of the multivariate analyses were conducted. First, larval branchiohyoideus and the adult su- a longitudinal design was used for the two barcualis rectus one and because of the dif- individuals for whom we had electromy- ficulty of reliably recording from the larval ographic data at all three stages (Table 1). SAR1, we present electromyographic data These data were analyzed with a two-way from both the larval BH and the adult SARl. (univariate) factorial analysis of variance This allows us to determine if nonhomolo- (ANOVA) (Sokal and Rohlf, '81) with met- gous muscles with similar functions possess amorphic stage as a fixed effect and individ- similar activity patterns. ual variance as a random effect. Second, a cross-sectional design was used for the 11 Experimental design and statistics individuals that were studied at one stage The goal of our experimental design was each (Table 1). These data were analyzed to test for mean differences in cranial muscle using a three-level (univariate) nested AN- activity patterns during 1)the environmen- OVA (Sokal and Rohlf, '81). Variance in each tal change from feeding in the water to feed- motor pattern variable is partitioned into ing on land, and 2) the morphological changes components attributable to metamorphic occurring at metamorphosis. In order to do stages, to individuals within a metamorphic this, we used the general plan of compari- stage, and to error variance as in previous sons outlined in Figure 1. The crucial aspect research (Lauder and Shaffer, '85; Shaffer of this experimental design is the ability of and Lauder, '85). We adopted the P 6 .01 metamorphosed Ambystoma tigrinum to feed level of significance rather than the normal underwater, as this allows us to test for dif- P s .05 to minimize the problem of finding ferences in the motor pattern with morpho- significant results only because of the fact logical and environmental shifts separately that many comparisons were being made si- (Fig. 1). Without this component of our ex- multaneously. perimental design, we would not be able to Approximately ten feedings were avail- separate changes in the motor pattern at able for each individual, although sample metamorphosis from those that occur with sizes varied among individuals: actual sam- the environmental transition from water to ple sizes are given with the summary sta- land. tistics for both the longitudinal and cross- In this paper we will refer to three onto- sectional data sets (Table 2). A total of 146 genetic stages. We are not strictly referring feedings were analyzed for this paper. to three distinct morphological units but Although correlations among the vari- rather to levels of comparison in the exper- ables were low (for example, with the cross- imental design. The three stages are 1) lar- sectional data set and a 19-by-19matrix, only val animals, which feed in the water, 2) me- 13 correlations out of 171 comparisons were tamorphosed animals feeding in the water, above 0.401, univariate analyses still may termed metamorphosed aquatic, and 3) me- provide an unreliable overall guide to mul- tamorphosed animals feeding on land, termed tivariate effects (Bray and Maxwell, '85; metamorphosed terrestrial (Fig. 1). Dunteman, '84; Harris, '75; Willig et al., '86). When we compare feedings between lar- Therefore, we used the largest data set (the val and metamorphosed aquatic animals, the cross-sectional one) to conduct a series of feeding behavior in both cases is aquatic suc- multivariate analyses. Because of the many tion feeding (Lauder, '85; Lauder and Shaf- variables used in our univariate analyses, fer, '86). These behaviors are similar in ki- we first chose a biologically meaningful sub- nematic pattern (Shaffer and Lauder, '88) set of eight variables, then used a principal but do differ in performance as documented components analysis to reduce further the by Lauder and Shaffer ('86). Test 1in Figure dimensionality of the data set to four factors. 1 thus tests for differences in the mean mo- The large number of variables and the rel- tor pattern before and after metamorphosis. atively small number of observations for each Comparing the metamorphosed aquatic stage individual (about ten) precluded a direct a to the metamorphosed terrestrial stage (Fig. priori multivariate analysis of variance 1, Test 2) tests for differences in the mean (MANOVA). The principal components PM

I I Fig. 4. Ambystoma tigrinum. Lateral view of cranial geniohyoideus muscle; D, dentary bone; DM, depressor osteology in a larval individual (A) and a metamor- mandibulae muscle; EP, epaxial muscles; H, humerus; phosed individual (B). Arrows indicate the direction of HB, hypobranchial cartilage; HH, hypohyal cartilage; hyoid depression during feeding. Scale bar = 1.0 cm. IH, interhyoideus muscle; IMP, intermandibularis pos- Abbreviations for this and subsequent figures: AME, terior muscle; MX, maxillary bone; PC, procoracoid car- adductor mandibulae externus muscle; AMI, adductor tilage; Pmectoralis muscle; PMX, premaxillary bone; mandibular internus muscle; BH, branchiohyoideus RC, rectus cervicis muscle; S, scapula; SAR1, subarcu- muscle; BHY, Basihyal cartilage; CB1-4, ceratobran- alis rectus 1 muscle; SC, scapular cartilage; SQ, squa- chial bones 1 through 4;CH, Ceratohyal cartilage; GH, mosal bone; UH, urohyal. analysis factored the correlation matrix of MANOVA analyses corresponded directly to eight variables (peak activity and duration tests 1 and 2 of Figure 1and utilize the same for the depressor mandibulae, epaxial, rec- experimental design and F-ratio construc- tus cervicis, and adductor mandibulae ex- tion as the univariate ANOVAs described ternus muscles) for 90 individual feeding above. trials. Four principal components were ex- tracted, and plots of the factor scores were Data analysis: techniques and variables examined €or patterns of dispersion among The motor pattern to the jaw muscles was stages. quantified by measuring the pattern of elec- Two nested MANOVAs were then con- trical activity produced by six individual ducted on the factor scores for components muscles during prey capture. Electromy- 1 and 2 (Chatfield and Collins, '80). The ographic (EMG) data were gathered from 256 G.V. LAUDER AND H.B. SHAFFER

TABLE 3. Two-way analysis of variance for 23 electromyographic (EMG) variables (longitudinal data set) measured from prey capture events (captures only) in Ambvstoma tierinum’ Metamorphic EMG stage Individual Interaction variable (2, 2) (1,50) (2, 50) DMMAX 1.0 0.8 8.4** DMTMAX 4.66 8.2* 4.4 DMDUR 6.3 2.7 4.7 EPON 5.0 0.1 4.0 EPMAX 1.6 1.4 4.3 EPTMAX 2.0 0.2 4.1 EPDUR 1.3 4.2 10.7** RCON 0.1 2.8 19.7~ RCMAX 5.1 2.0 6.2* RCTMAX 2.0 10.5* 4.9 RCDUR 2.0 0.1 15.4**

AMEON~~ ~~ ~ ~ 0.3 0.6 4.4 AMEMAX 3.6 2.6 4.6 AMETMAX 1.1 20.8** 22.2** AMEDUR 1.2 19.6** 17.0** AMION 0.1 62.7** 19.8** AMIMAX 0.1 1.7 5.6% AMITMAX 30.3 1.0 0.2 15.1 0.2 1.1 44.5 1.9 0.2 5.3 1.4 5.1* 67.5 0.2 0.2 BHiSARlDUR 2.0 6.7 15.7** ‘Entries in the table are F-values: The degrees of freedom for each test are given below the effect. Variable descriptions are given in the text. *P s .01. **P s ,001.

TABLE 4. Three-level nested analysis of variance for 23 electromyographic (EMG) variahles (cross-sectional data set) measured from prey capture events (captures only) in Ambystoma tierinurn‘ Among Among individuals metamorphic within a Among feedings EMG stages metamorphic stage within v a r i ab 1e (2, 8) (8, 79) individuals DMMAX 18 42 DMTMAX 3 91 DMDUR 15 63 EPON 0 73 EPMAX 0 29** 71 EPTMAX 44* 13* 43 EPDUR 21 22** 57 RCON 33 11 56 RCMAX 0 52** 48 RCTMAX 8 26** 66 RCDTJR-~. 41 33** 26 AMEON 59 * 16%’” 25 AMEMAX 14 33** 53__ AMETMAX 20 13 67 AMEDUR 9 26** 65 AMION 0 51** 49 AMIMAX 49* 12* 39 AMITMAX 13 23 * 64

21~~ 5 74 51 21** 28 62 23** 15 25* 0 75 BHiSARlDUR 1 24* 75 ‘Values represent variance components (in %); Degrees of freedom for stage and individual effects are shown. Variable descriptions are given in the text. *P = .01. *:T = ,001. ONTOGENY OF MOTOR PATTERNS 257 each individual using techniques similar to file (data matrix) consisting of six columns, those practiced previously (Lauder and one for each muscle, and 5,096 rows corre- Shaffer, '85; Wainwright and Lauder, '86). sponding to the 4 seconds that each analog Bipolar stainless steel electrodes (0.051 mm feeding was sampled. This digital file was in diameter) were implanted percutaneously then run through a program that measured into six muscles of the head. Bared electrode a total of 23 variables from the muscle ac- tips were approximately 0.5 mm long, and tivity pattern. Uniform settings for baseline the electrode tips were about 1 mm apart in noise and the detection of activity in a mus- the muscle. Electrode pairs were glued to- cle were used for all analyses, thus avoiding gether and were often sutured to the skin to any subjective component in the determi- minimize displacement. All six pairs of elec- nation of muscle onset and offset times. This trodes were glued together and sutured to program used the onset of activity in the the back of the animal. depressor mandibulae muscle as a reference EMG signals were amplified 5,000-10,000 from which to determine when other mus- times using Grass P511J preamplifiers and cles started their electrical activity. As in a bandpass of 100-3,000 Hz. The signals were previous research (Lauder and Shaffer, '851, recorded on a Bell and Howell 4020A FM the depressor mandibulae was chosen as a tape recorder for future analysis. reference because of its consistent and high Quantitative analyses of variation in level of activity and its role as a major mouth muscle activity among individuals and among opening muscle. electrode placements within a muscle have Twenty-three variables were measured by been presented elsewhere (Lauder and Shaf- the computer program from the digital file fer, '85; Shaffer and Lauder, '85):we have of each feeding (variable abbreviations given not found differences in electrode placement here correspond to those used in the tables to be a significant source of variation in our and throughout this paper). The onset of ac- data. tivity in each muscle (in ms) was measured The motor pattern to six muscles was re- relative to the onset of activity in the de- corded simultaneously: the depressor man- pressor mandibulae (five variables total): dibulae (DM), the epaxial muscles (EP), the EPON, the onset of activity in the epaxial rectus cervicis (RC), the adductor mandi- muscles; RCON, the onset of activity in the bulae internus and externus (AM1and AME), rectus cervicis muscle; AMEON, the onset and the branchiohyoideus (BH, in larvae) of activity in the adductor mandibulae ex- and the subarcualis rectus one (SAR1, in ternus muscle; AMION, the onset of activity metamorphosed animals). These muscles in the adductor mandibulae internus mus- were chosen because 1)they cover all major cle; BH/SARlON, the onset of activity in the functional components of the head (mouth branchiohyoideus (in larvae) or the subar- opening, mouth closing, head elevation, hyoid cualis rectus one muscle (in adults). depression, hyoid elevation); 2) they can be The duration of electrical activity in each homologised across metamorphosis; and 3) muscle (in ms), the maximum (rectified) am- they are present through ontogeny. Two other plitude of activity in each muscle (in volts), possible sets of muscles that might have been and the time from the onset of activity to studied are the intrinsic tongue muscles and peak amplitude (in ms) were also measured the muscles of the gill arches. We did not for a total of three variables for each muscle: record from these muscles because the ton- respectively, depressor mandibulae gue muscles are acquired at metamorphosis (DMDUR, DMMAX, DMTMAX); epaxial and the gill arch muscles are lost at meta- muscles (EPDUR, EPMAX, EPTMAX); rec- morphosis. These muscle groups are thus in- tus cervicis (RCDUR, RCMAX, RCTMAX); appropriate for the study of motor pattern adductor mandibulae externus (AMEDUR, conservatism across a major morphological AMEMAX, AMETMAX); adductor mandi- change. bulae internus (AMIDUR, AMIMAX, Electromyographic data from each feed- AMITMAX); branchiohyoideus/subarcualis ing were played from the FM tape into a 12- rectus one (BH/SARlDUR, BHISARlMAX, bit analog-to-digital converter sampling each BH/SARlTMAX). channel at 1,274 Hz. These data were then The goal of these measurements from each stored on a hard disc for subsequent analy- feeding was to characterize completely and sis. Each feeding resulted in a digital data quantitatively the motor pattern in these six 258 G.V. LAUDER AND H.B. SHAFFER

BHY A / YHH

Fig. 5. Ambystoma tigrinurn. Ventral views of the tobranchials 3 and 4 and the alterations in hyoid and hyobranchial apparatus in a larval individual (A) and basibranchial shape at metamorphosis. Scale bar = 1.0 a metamorphosed individual (B). Note the loss of cera- cm. For abbreviations, see legend to Figure 4. jaw muscles to allow us to determine pre- reabsorbed. The eyes change from an an- cisely which aspects (if any) of the pattern terolateral orientation located deeply in eye change during ontogeny. sockets in the skull to a more anterior orientation raised up off the surface of the head RESULTS (Figs. 2, 3). As the external gills are being Morphology reabsorbed, the gular membrane, which has General a free posterior margin in larvae, attaches The morphology of both larval and adult and fuses to the skin of the body wall. In ambystomatid salamanders has been de- metamorphosed individuals there is no con- scribed in some detail in the literature (Bo- nection posteriorly from the buccal cavity to nebrake and Brandon, '71; Carroll and the exterior. In terms of feeding mecha- Holmes, '80; Edgeworth, '35; Jarvik, '63; nisms, the process of aquatic feeding has been Krogh and Tanner, '72; Larsen and Guthrie, converted from a unidirectional flow system '75; Latimer and Roofe, '64; Lauder and in which water enters the mouth and leaves Shaffer, '85, '86; Luther, '14; Piatt, '38, '39, via the gill slits and gular membrane to a '40; Regal, '66; Reilly, '87). However, these bidirectional system in which water must works do not provide a complete description both enter and leave the buccal cavity via of ambystomatid morphology in the context the mouth (Lauder and Shaffer, '86). of metamorphosis and functional analysis, and we present additional information here Osteology and myology to aid in the interpretation of changes in the Equally striking modifications occur in the motor pattern during ontogeny. musculoskeletal system of the head. Cera- tobranchials 3 and 4 are lost completely at Basic design features metamorphosis (Figs. 4'5: CB) as is the me- As metamorphosis proceeds in Ambys- dian cartilaginous connection of the urohyal toma tigrinurn, striking changes occur in the to the basihyal. Major changes in hyoid and external morphology of the head (Figs. 2'3). branchial arch shape occur. The large lat- The head becomes shallower and narrower eral mass of ceratobranchials visible bein lateral view, and the external gills are tween the pectoral girdle and squamosal in ONTOGENY OF MOTOR PAlTERNS 259 A EP

Fig. 6. Ambystoma tigrinurn. Lateral (A) and ven- the left side to show the deeper musculature. Scale tral (B) views of the cranial musculature in a larval bar = 1.0 cm. For abbreviations, see legend to Figure individual. The ventral throat muscles (interhyoideus 4. and intermandibularisposterior) have been reflected on 260 G.V. LAUDER AND H.B. SHAFFER

TABLE 5. Loadings of eight variables on principal components 14(PCI -4)' Component loadings EMG variable PC 1 PC2 PC3 PC4

DMMAX - .76 - ,051 .09 - .05 DMDUR - .75 .38 .10 .09 AMEMAX - .58 - .35 .12 52 RCMAX - 50 .38 - .39 - .34 EPDUR .06 .70 .45 .04 RCDUR .06 .65 - 57 - .05 AMEDUR .26 .57 .06 .63 EPMAX .01 .20 .75 - .38 Variance 22.6% 21.2% 16.3% 11.8% ex D 1a i n ed 'Factor scores for prey captures on components one and two are plotted in Figure 10. The variance explained by each of the first four components is given (in %) below each column. Variable descriptions are given in the text. larvae (Fig. 4A) is greatly reduced in size BH/SARlTMAX) suggest that larger sam- after metamorphosis (Fig. 4B). The toothed ple sizes might reveal these to be significant margin of the gape increases, and a fleshy differences among metamorphic stages. Of tongue pad forms in the buccal cavity dorsal these variables with large F-values, the to the basihyal. means for the two adductor mandibulae in- The lines of action and major muscle masses ternus variables are most different for the change relatively little at metamorphosis terrestrial stage (Table Z), indicating that (Figs. 6,7).The muscles spanning the man- no change in the motor pattern accompanied dibular rami ventrally are the intermandi- the morphological changes at metamorpho- bularis posterior and interhyoideus. This sis in these variables. Terrestrial feedings latter muscle lies in the posterior margin of exhibited substantially longer duration ad- the gular flap in larvae (Fig. 6: IH). The gen- ductor mandibulae internus activity than did iohyoideus connects the urohyal and man- either of the two aquatic stages. The mean dibular symphysis. Both jaw adductor mus- for the variable with the highest F-value, cles maintain their relative positions through BWSARlTMAX (Table 2) is most different ontogeny, although the adductor mandibu- for larvae, indicating that the SARl muscle lae externus has a slightly more extensive in metamorphosed individuals has a differ- insertion on the dentary after metamorpho- ent time to maximum activity than the sis (Figs. 6, 7: AME). branchiohyoideus (BH) muscle in larvae. The Following metamorphosis the depressor two individuals studied proved to be differ- mandibulae acquires a fan-shaped origin from ent primarily in the adductor muscle vari- the skull and looses the fibers that inter- ables, as shown by the significant individual mingle with the fibers of the branchiohyoi- effects in Table 3. deus. After metamorphosis the subarcualis The cross-sectional data set (Table 4) has rectus one muscle extends from the first cer- much greater statistical power to test for an atobranchial to the ceratohyal, and the lar- effect of metamorphic stage. In the univar- val branchiohyoideus muscle is lost. iate analyses, four variables were found to have a significant proportion of variance as- The motor pattern sociated with stage (Table 4). Three of these Summary statistics for the 23 variables in (EPTMAX, AMIMAX, and BH/SARlTMAX) each of the two data sets are presented in involve maximum voltages. Comparisons Table 2. Representative myograms from one among the nonhomologous BH and SARl individual Ambystoma tigrinum at three on- muscle variables showed that larvae are sig- togenetic stages are shown in Figure 8. nificantly different from other stages in the In the longitudinal data set none of the 23 time to maximum activity (Table 4: BH/ variables show a significant effect of meta- SARlTMAX). Similarly, larvae are most morphic stage (Table 3). In this experimen- different from other stages in the maximum tal design our samples sizes are low, and the amplitude of electrical activity (Table 4: BH/ high F-values for four of the variables SARlMAX), a variable having a high (but (AMITMAX, AMIDUR, BH/SARlON, and not significant) among-stage variance com- ONTOGENY OF MOTOR PATTERNS 261

PE B

. PE

IMP

Fig. 7. Ambystoma tigrinum. Lateral (A) and ven- reflected on the left side to show the deeper musculature. tral (B) views of the cranial musculature in a meta- Scale bar = 1.0 cm. For abbreviations, see legend to morphosed individual. The ventral throat muscles (in- Figure 4. terhyoideus and intermandibularisposterior) have been ponent. These variables reflect changes in variables were most different in larvae, in- function between the nonhomologous bran- dicating a change in motor pattern in the chiohyoideus and subarcualis rectus mus- time to maximal epaxial muscle activity and cles during ontogeny. Two of the significant the amplitude of activity in the adductor 262 G.V. LAUDER AND H.B. SHAFFER

Metamorphosed - terrestrial Larval Metamorphosed - aquatic

.E P

”+--.---

AM1

AME < 1 -.* t ..F

100 ms Fig. 8. Representative electromyographic (EMG) was plotted. The vertical scale is in millivolts. The motor traces from one individual Ambystoma tigrinurn to show pattern was found not to be significantly different be- the motor pattern at three ontogenetic stages. These tween the larval and the metamorphosed aquatic stages. data were converted from analog to digital as discussed For abbreviations, see legend to Figure 4. in “Materials and Methods,” and then this digital file mandibulae internus (Table 4: EPTMAX, principal components 1 and 2 (Table 5: PC1 AMIMAX). Among individuals, variance was and PC2). An examination of the component significant in 18 out of the 23 total variables, loadings shows that high PC1 scores are de- indicating a high degree of heterogeneity termined by negative loadings of depressor among individuals within stages. mandibulae amplitude and duration and by A summary bar diagram of the pattern of negative loadings of the amplitude of activ- electrical activity in the six muscle groups ity in the rectus cervicis and adductor man- for the longitudinal data set is shown in Fig- dibulae externus. Principal component two ure 9. Variation in the offset of muscle ac- reflects positive loadings of the duration of tivity is generally greater than in the onset, activity in the epaxial muscles, rectus cer- and onset times are very similar to each other. vicis, and the adductor mandibulae externus There is little consistent variation in the (Table 5). pattern of muscle duration with ontogenetic The multivariate analysis of variance stage. (MANOVA) comparing the mean factor Multivariate dispersion of the motor pat- scores of larval and metamorphosed aquatic tern among the three ontogenetic stages is feedings (test 1 in Fig. 1) on the first two illustrated in Figure 10, and the loadings on principal components showed no significant components 14are given in Table 5. Aquatic differences (P = .136, DF = 2, 4). In con- feedings (a comparison of larval and meta- trast, the MANOVA comparing metamor- morphosed aquatic feedings) are quite sim- phosed aquatic and terrestrial feedings (test ilar to each other with broadly overlapping 2, Fig. 1)was significant at P = .014 (DF = 2, distributions on the first two principal com- 4). Discriminant function analysis correctly ponents (which together account for 43.8% classified 84% of the larval feedings and only of the variance). Terrestrial feedings, how- 62% of the metamorphosed aquatic feedings ever, have distinctly high values on both in the test 1 comparison, while 92% of both ONTOGENY OF MOTOR PAlTERNS 263 metamorphosed aquatic and terrestrial Although univariate analyses did not show feedings were correctly classified in the test a modification of the motor pattern with the 2 comparison. transition from water to land (test 2 in Fig. 1; Tables 3,4), the multivariate analysis (of DISCUSSION the cross-sectional data set) revealed that Are motor patterns conserved during on- metamorphosed terrestrial individuals do togeny? Our data show clearly that the mean have distinctive muscle activity patterns. motor pattern to the six jaw and hyoid mus- Thus, the MANOVA shows that the cen- cles does not change with metamorphosis. troids for the metamorphosed aquatic and Thus, the motor pattern is conserved across terrestrial polygons in Figure 10 are signif- this major morphological transformation. icantly different. Terrestrial feedings tended Four aspects of our data and analysis sup- to have longer activity durations in the jaw port this interpretation. (It is important to muscles and lower amplitude activity in the remember that comparing mean motor pat- rectus cervicis and adductor mandibulae ex- terns across a morphological transformation ternus. is test 1 in the experimental plan of Fig. 1.) The differences between aquatic and ter- First, very few of the 23 variables in the restrial feedings could be due to changes in univariate analyses (Tables 3,41 show a sta- the hydrodynamic nature of the environ- tistically significant stage effect. Second, ment alone. Because air is less viscous and there is virtually no congruence between the dense than water, one might expect that, longitudinal and cross-sectional analyses in ceterisparibus, feedings in air should be more the few variables that are significant. As the rapid than in water because of the reduced statistical power of our two-way ANOVA was resistance, and that peak bone excursions limited by the fact that we were able to ob- should be reached more quickly. Kinematic tain longitudinal recordings from only two data indicate that in fact the converse is true individuals, one might wish to examine all (Shaffer and Lauder, '881, and the electro- variables having large F-values, regardless myographic data support this result. Ter- of whether these are statistically significant restrial feedings involve longer-duration or not. If there is congruence among the two muscle activities (Tables 2, 5; Fig. 10) and analyses in variables with high F-values or there is no evidence of a purely physical ef- variance components,that would suggest that fect on the motor pattern. there really is a change in the motor pattern However, the change from aquatic to ter- that our statistical analysis failed to detect restrial motor patterns might well be due to as significant. This is not the case for any of sensory feedback. The change in motor pat- the homologous muscles (Tables 3,4).Third, tern between metamorphosed aquatic and the level of intercorrelation among the vari- terrestrial individuals need not involve the ables studied was low, and there was little use of different feeding circuits, but could congruence among the two data sets in the represent modulation of a single feeding cir- structure of the correlation matrices. No cuit based on environment. Terrestrial feed- variables emerged as being consistently ing involves tongue projection toward the highly correlated with each other. Fourth, prey (Shaffer and Lauder, ,881, which is the principal components analysis (Fig. 10) achieved with muscles acquired at meta- shows considerable overlap between larval morphosis. The tongue projection feeding be- and metamorphosed aquatic stages, and the havior with the concomitant longer duration MANOVA comparing these feedings was not muscle activities could well be activated by significant. The MANOVA effectively tests sensory inputs from skin mechanoreceptors the centroids of the two aquatic polygons in or by the sensation of gravity. Figure 10 to determine if they are signifi- The lack of significant mean differences cantly different. As we have examined mus- in the motor pattern between pre- and post- cles that control all major aspects of head metamorphic Ambystoma tigrinum bears on function that are present throughout ontog- previous hypotheses of motor pattern con- eny, we conclude that there is no evidence servatism in vertebrates (e.g., Bramble and that the mean motor pattern changes at Wake, '85; Byrd, '85; Goslow, '85). There is metamorphosis when the environment is held no a priori reason why motor patterns should constant. be conserved across major environmental and However, there is a multivariate enuiron- morphological changes in ontogeny and phy- mental effect on the motor pattern (Fig. 10). logeny. It is possible to adduce evidence both 264 G.V. LAUDER AND H.B. SHAFFER Bl DM EP - RC 1.11) I .-- AMEI .-- I AM1 I SARl I

I, I 11I I A L. i I i,1 I11 1 I1 - 0 40 80 120 0 100 200 TIME (in ms) CI TIME (in ms) DM -

1-k AM1 1-k SARl I- I _L.LLYuUJ 40 80 120 ' iio ' ioiii TIME (in ms)

Fig, 9. Bar diagrams summarizing the EMG pattern summary and confounds variation due to individuals in larval (A), metamorphosed aquatic (B), and terra- and feedings. This figure is thus useful for an overall trial (C) stages. The start and end of each bar indicate view of the pattern of muscle duration during ontogeny the mean onset and offset of activity for each muscle. but cannot be used for statistical comparisons of the The thin lines extending horizontally from the bars rep- ontogenetic stages (see Tables 3 and 4 for these results). resent 1 standard deviation of the mean onset and offset For abbreviations, see legend to Figure 4. times. Note that this figure presents only an overall

in support of the view that the motor pattern suckling behavior in mammals (Byrd, '85; should not change as well as in support of Herring, '85; Moyers, '73; Sessle, '76). Anal- the idea that morphological and environ- yses of trigeminal motor neuron populations mental shifts should be accompanied by sig- in two vertebrates, frogs and lampreys (Al- nificant changes in the average motor pat- ley and Barnes, '83; Barnes and Alley, '83; tern. In support of conservatism, many Homma, '78), have shown that motoneuron aspects of central nervous system structure populations tend to be conserved across mor- are similar across vertebrate lineages (e.g., phological changes occurring in metamor- Northcutt, '77, '78; Sarnat and Netsky, '741, phosis. (Note that these last studies actually and such similarities may reflect a corre- show that motoneuron morphology and rel- sponding conservatism of motor output to ative position tends to be conserved, not the peripheral musculature. In invertebrates, motor output from the neural circuits.) neuronal circuitry may be conserved across On the other hand, motor patterns are the species, even when peripheral muscles have product of neural circuitry that may change been lost (Arbas, '83; Dumont and Robert- both during ontogeny and between species son, '86). In vertebrates, key proposed ex- (Bush and Clarac, '85; Ewert, '76; Laming, amples of motor pattern conservatism have '81; Levine and Truman, '82; Technau and included analyses of limb muscle motor pat- Heisenberg, '82). Even quite similar simple terns in chicks (Bekoff,'76; Bekoff and Kauer, behaviors may be produced by very different '84) and the development of mastication from neural circuits (Dumont and Robertson, '86; 2.5 I I I I I

1.5

0.5

-0.5

-1.5

-2.5 -2.5 -1.5 -0.5 0.5 1.5 2.5

FACTOR 2 Fig. 10. Principal components analysis on 90 feed- two (and the polygon centroids are not significantly dif- ings and eight variables (see text for details) to illustrate ferent, MANOVA P = .136),but the polygon centroid the multivariate relationship among the motor patterns for terrestrial feedings is significantly different from that for the three ontogenetic stages. Each point represents for metamorphosed aquatic prey captures (MANOVA one feeding. Larval and metamorphosed aquatic feed- P = ,014). ings overlap broadly on principal components one and

Lauder, '86), and it is clear that new func- Mechanisms of behavioral transformation tional specializations may indeed be corre- The results presented above on motor pat- lated with changes in the motor pattern. The terns in Ambystoma tigrinum now need to work of Graf and Baker ('83, '85a,b) has shown be considered in light of the three-part re- elegantly that changes in neuronal circuitry search program for the analysis of behav- accompany metamorphosis in flatfish (Pseu- ioral transformation outlined in the opening dopleuronectes). During ontogeny, behav- section. ioral changes also may be accompanied by First, what are the behavioral changes (if modification of motor patterns (Bentley and any) that occur during ontogeny for which Hoy, '701, and the anatomical basis of motor we would like to determine a mechanistic systems may vary considerably from species basis? Mean changes in feeding behavior or to species. Also, metamorphosis may involve performance have been documented for both alterations in neuronal populations and re- test 1and test 2 comparisons (Fig. 1).Lauder organization of dendritic fields (Casaday and and Shaffer ('86) showed that there was a Camhi, '76; Taylor and Truman, '74; Tru- significant decrease in performance by me- man and Reiss, '76; Truman et al., '851, im- tamorphosed Ambystoma tigrinum feeding plying that neuronal circuits have changed. in the water when compared with larval per- Fetcho ('86a,b) has shown that the organi- formance. Reilly and Lauder ('88a) docu- zation of motoneuron pools innervating ax- mented similar significant performance de- ial musculature in aquatic vertebrates such creases in Notophthalmus viridescens at as fishes and nontransforming salamanders metamorphosis. In addition, Shaffer and is very different from that of terrestrial ver- Lauder ('88) have documented mean changes tebrates. Thus, the central neuronal organ- in feeding kinematics by Ambystoma ti- ization containing the circuits that produce grinum between terrestrial and aquatic me- motor patterns to the axial muscles in ver- tamorphosed individuals. Thus, there are tebrates is not phylogenetically conserva- significant behavioral changes for both com- tive. 266 G.V. LAUDER AND H.B. SHAFFER parisons outlined in Figure 1. a significant change in the motor pattern Second, do correlated changes occur in (Tables 3, 4). Do the changes in the motor mean morphology or physiology during on- pattern account for the alteration in behav- togeny that might serve to explain any sig- ior? Although no direct (manipulative) ex- nificant mean behavioral differences? The perimental evidence exists on this point, the results of this study show that the lines of biomechanics of the feeding system strongly action and basic design of the musculoskel- indicate that the observed changes in motor etal system remain relatively unchanged pattern in test 2 do account for the observed throughout ontogeny. However, there is a behavioral transformation. One of the key major change at metamorphosis in several distinguishing features of terrestrial feed- features of the head: the gill slits close, and ing kinematics in Ambystoma tigrinum is the head changes from a unidirectional to a the longer duration in nearly all kinematic bidirectional feeding system (Figs. 2, 3). AS variables measured: terrestrial feedings take documented above, there is no change in the longer to complete than aquatic feedings mean motor pattern at metamorphosis (Fig. (Shaffer and Lauder, '88). The motor pattern 1: test 1; Tables 3, 41, but there is a signif- shows a similar trend, with terrestrial feed- icant change in the mean motor pattern used ings exhibiting longer-duration muscle ac- for feeding in the two environments (Fig. 1: tivities (Fig. 10; Table 5: high loadings of test 2; Tables 3, 4). the duration of activity in the epaxialis, rec- Third, is there any evidence that the cor- tus cervicis, and adductor mandibulae ex- related changes in form or function account ternus). Thus, as one might expect biome- for the observed changes in behavior in each chanically, longer feedings are associated of the two tests? For test 1, we observed no with longer durations of muscle activity. significant change in the motor pattern nor any major alteration in musculoskeletal de- CONCLUSIONS sign. Both of these factors can thus be elim- The issue of conservative muscle activity inated as possible causal bases of ontoge- patterns is an important one in functional netic alterations in feeding performance. We morphology because it directly affects how therefore hypothesize that it is the change one explains behavioral and functional in functional design of the head (from uni- transformations in animals. Is behavioral directional to bidirectional) that causes the transformation a function of alterations in decrease in feeding performance during on- the motor pattern, peripheral morphology, togeny. Lauder and Shaffer ('86) showed that or both? The data presented here and pre- the metamorphosis of the feeding mecha- vious research on vertebrate feeding sys- nism from unidirectional to bidirectional flow tems (Byrd, '85; Herring, '85; Lauder, '83, has a significant effect on the pattern of pres- '85; Wainwright and Lauder, '86) indicate sure generated in the mouth cavity of tiger that motor patterns are conserved during the salamander. Lauder and Reilly ('88) con- origin, either in ontogeny or phylogeny, of ducted a series of manipulatiue experiments behavioral novelties. How general might that examined buccal pressures and feeding these results be? Are novel behaviors gen- performance in axolotls (Ambystoma mexi- erally produced by modifications of the mus- canum) in which the gill openings had been culoskeletal system or might motor patterns sutured closed. These experiments tested the be changed also (Lauder, '88)? There is still hypothesis that experimental closure of the far too little quantitative evidence to gen- gill slits alone (with no change to the mus- eralize on this issue, but it is possible to sug- culoskeletal system) would produce a sig- gest several future research directions that nificant decrease in buccal pressure and will help resolve the question. First, more feeding performance compared to control an- case studies explicitly and quantitatively imals. This indeed was the result. Thus, the comparing motor patterns across major mor- hypothesis that the causal basis for onto- phological and environmental changes are genetic changes in behavioriperformance is needed. Are environmental changes neces- the change in gross design of the skull and sarily associated with alterations in the mo- not differences in motor pattern is corrobo- tor pattern? Second, comparative phyloge- rated. netic analyses are needed on motor pattern For test 2, there is a documented behav- transformation in relation to changes in ioral transformation (Shaffer and Lauder, morphology and behavior. The mapping of ,881, no change in morphology (Fig. 11, and behavioral, morphological, and motor pat- ONTOGENY OF MOTOR PATTERNS 267 tern characters onto a phylogeny will enable analysis. Science 170:1409-1412. us to ascertain quantitatively the degree of Bonebrake, J.E., and R.A. Brandon (1971) Ontogeny of cranial ossification in the small-mouthed salamander, concordance in transformation of these three Ambystornu texunum. J. Morphol. 133:189-204. types of characters. Third, comparative Bramble, D.M., and D.B. Wake (1985)The feeding mech- analyses of differing types of behaviors are anisms of lower tetrapods. In M. Hildebrand, D.M. needed to assess the generality of results on Bramble, K.F. Liem, and D.B. Wake (eds): Functional Vertebrate Morphology. Cambridge: Harvard Uni- feeding systems. Ballistic behaviors, such as versity Press, pp. 230-261. most aquatic feeding by lower vertebrates, Bray, J.H., and S.E. Maxwell (1985) Multivariate Anal- may be associated with conserved motor pat- ysis of Variance. Beverly Hills: Sage Publications. terns, whereas behaviors requiring signifi- Bush, B.M.H., and F. Clarac (1985) Coordination of Mo- tor Behavior. Cambridge: Cambridge Univ. Press. cant sensory feedback during their execu- Byrd, K.E. (1985) Research in mammalian mastication. tion may not show conserved motor patterns. Am. Zool.25:365-374. By integrating results from ontogenetic and Carroll, R.L., and R. Holmes (1980) The skull and jaw phylogenetic analyses, perhaps a general musculature as guides to the ancestry of salamanders. Zool. J. Linn. SOC.Lond. 68:140. picture of the relationship between behavior Casaday, G.B., and J.M. Camhi (1976) Metamorphosis and its morphological and neural bases will of flight motor neurons in the moth Manduca sexta. emerge. J. Comp. Physiol. A. 112:143-158. Chatfield, C., and A.J. Collins (1980) Introduction to ACKNOWLEDGMENTS Multivariate Statistics. London: Chapman and Hall. Dingerkus, G., and L.D. Uhler (1977) Enzyme clearing We thank Peter Wainwright and Steve of alcian blue stained whole small vertebrates for Reilly for their helpful comments on this demonstration of cartilage. Stain Technol. 52:229-232. manuscript and for discussions and joint re- Druner, L. (1902) Studien zur Anatomie der Zungen- bein-, Kiemenbogen-, und Kehlopfmuskelen der Uro- search in functional morphology. Conver- delen. I Theil. Zool. Jahrb. Anat. 15:435-622. sations with Ted Goslow, Willy Bemis, Eric Duellman, W.E., and L. Trueb (1986) Biology of Am- Findeis, Dave Wake, Gerhard Roth, and Ken phibians. New York: McGraw Hill. Dial were instrumental in formulating our Dumont, J., and R.M. Robertson (1986) Neuronal circuits: An evolutionary perspective. Science 233t849- ideas on motor patterns. The anatomical fig- 853. ures were drawn by Clara Richardson. The Dunteman, G.H. (1984) Introduction to Multivariate Ambystoma tigrinum larvae were kindly Analysis. Beverly Hills: Sage Publications. collected by Tom Kocher. The computer pro- Edgeworth, F.H. (1935) The Cranial Muscles of Verte- brates. Cambridge: Cambridge Univ. Press. gramming so necessary to this project was Emerson, S., and D. Diehl (1980) Toe pad morphology done superbly by Cathy Smither and Dojun and mechanisms of sticking in frogs. Biol. J. Linn. Yoshikami. This research was supported by SOC.Lond. 13:199-216. NSF DCB 86-02606, DCB 87-21010, and NSF Ewert, J.-P. (1976) Neuroethology. Springer Verlag: New York. BSR 85-20305 to G.L., and NSF BSR 85- Fetcho, J.R. (1986a) The organization of the motoneu- 19211 to H.B.S. rons innervating the axial musculature of vertebrates. I. Goldfish (Carussius aurutus) and mudpup- LITERATURE CITED pies (Necturus maculosus).J. Comp. Neurol. 249521- Alley, K.E., and M.D. Barnes (1983) Birth dates of tri- 550. geminal motoneurons and metamorphic reorganiza- Fetcho, J.R. (1986b) The organization of the motoneu- tion of the jaw myoneural system in frogs. J. Comp. rons innervating the axial musculature of verte- Neurol. 218:395-405. brates. 11. Florida water snakes (Nerodiafasciata pic- Arbas, E.A. (1983) Neural correlates of flight loss in a tiuentris). J. Comp. Neurol. 249.551-563. Mexican grasshopper, Barytettiz psolus. I. Motor and Francis, E.T.B. (1934) The Anatomy of the Salamander. sensory cells. J. Comp. Neurol. 216:369-380. London: Oxford Univ. Press. Arnold, S.J. (1983) Morphology, performance, and fit- Graf, W., and R. Baker (1983) Adaptive changes of the ness. Am. Zool. 23:347361. vestibuloocular reflex in flatfish are achieved by re- Barnes, M.D., and K.E. 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