JOURNAL OF MORPHOLOGY 17457-77 (1982)

Append icuI ar Skeletal Mor pho I og y in Mi nut e , Genus (Amphibia: ): Growth Regulation, Adult Size Determination, and Natural Variation JAMES HANKEN Museum of Vertebrate Zoology and Department of Zoology, University of California. Berkeley, California 94720 ABSTRACT Patterns of growth and variation of the appendicular skeleton were examined in Thorius, a speciose genus of minute terrestrial plethodontid sal- amanders from southern Mexico. Observations were based primarily on ontoge- netic series of each of five species that collectively span the range of adult body size in the genus; samples of adults of each of seven additional species provided supplemental estimates of the full range of variation of limb skeletal morphology. Limbs are generally reduced, i.e., pedomorphic, in both overall size and develop- ment, and they are characterized by a pattern of extreme variation in the composi- tion of the limb skeleton, especially mesopodial elements, both within and between species. Fifteen different combinations of fused carpal or tarsal elements are vari- ably present in the genus, producing at least 18 different overall carpal or tarsal ar- rangements, many of which occur in no other plethodontid genus. As many as four carpal or tarsal arrangements were observed in single population samples of each of several species; five tarsal arrangements were observed in one population of T. minutissimus. Left-right asymmetry of mesopodial arrangement in a given speci- men is also common. In contrast, several unique, nonpedomorphic features of the limb skeleton, in- cluding ossification of the typically cartilaginous adult mesopodial elements and ontogenetic increase in the degree of ossification of long bones, are characteristic of all species and distinguish Thorius from most related genera. They form part of a mechanism of determinate skeletal growth that restricts skeletal growth after sexual maturity. Interspecific differences in the timing of the processes of appen- dicular skeletal maturation relative to body size are well correlated with interspe- cific differences in mean adult size and size at sexual maturity, suggesting that shifts in the timing of skeletal maturation provide a mechanism of achieving adult size differentiation among species. Processes of skeletal maturation that confer determinate skeletal growth in Thorius are analogous to those typical of most amniotes - both groups exhibit on- togenetic reduction and eventual disappearance of the complex of stratified layers of proliferating and maturing cartilage in long bone epiphyses - but, unlike most amniotes, Thorius lacks secondary ossification centers. Thus, the presence of sec- ondary ossification centers cannot be used as a criterion for establishing determi- nate skeletal growth in all vertebrates.

Phyletic size change is a conspicuous feature opment of the skeleton - particularly the ap- of evolution, and the phenomenon of size in- pendicular skeleton - have figured prominently crease or decrease is well documented for many in attempts to explain growth regulation over- taxa (Stanley, '80). Less is known of the physi- all. Early development of the appendicular ological mechanisms of growth regulation by skeleton is remarkably similar in all Recent which size are achieved' In tetra- Dr. Hanken's current address is Department of Biology, Dalhousie pod vertebrates, patterns of growth and devel- University, HaMax B3H 4Jl.Nova Scotia, Canada

0362-2525/82/1741-0057$06.000 1982 ALAN B. LISS, INC. 58 J. HANKEN tetrapods; many differences among groups be- brates, the variation and distribution of these come apparent only at later stages (Hinchliffe mechanisms among different taxa, and the and Johnson, ’80). A typical long bone initially consequences of size change in particular forms as a mesodermal condensation that sub- groups. sequently differentiates into a cartilaginous The genus Thorius (Amphibia: Plethodonti- model, or precursor, of the later bony element. dae) comprises a complex of at least 15 species All tetrapods, as well as bony fishes, develop a of terrestrial lungless salamanders that live in single ossification center, or diaphysis, in the the montane forests of southern Mexico. Rep- midsection (shaft)of each long bone between resenting a specialized component of the ex- the two cartilaginous ends, or epiphyses tensive radiation of plethodontid salamanders (Haines, ’42). This single, or primary, ossifica- in the New World tropics, Thorius is highly de- tion center is typical of , turtles, rived in many respects, particularly in mor- and crocodilians, whereas mammals, birds, and phology, compared to related genera (Wake, the remaining Recent, limbed reptiles - liz- ’66). The most conspicuous attribute of Thor- ards and Sphenodon (thetuatara) - frequently ius is small size - as adults, body size ranges form additional, secondary ossification centers from as little as 14 mm SVL (snout to posterior in the cartilaginous epiphyses after the appear- end of vent)in the smallest species, tojust over ance of the primary center. During ontogeny in 30 mm SVL in the largest species. Associated these latter groups, primary and secondary os- with reduced size are several extreme modifica- sification centers increase in size relative to the tions of the appendicular skeleton. The limbs overall length of the bone and gradually erode are relatively small and the digits (especially the intervening cartilaginous plates that sepa- the terminal phalanges) are poorly developed; rate them. Because this cartilage is the only in these aspects, skeletal development is trun- tissue capable of longitudinal expansion (via cated precociously relative to that of more gen- cell proliferation and interstitial growth), lon- eralized plethodontid salamanders, conform- gitudinal bone growth is no longer possible ing to an overall pattern of pedomorphosis when primary and secondary centers meet and exhibited by much of the skeleton, including fuse. Consequently, secondary ossification the skull. In contrast, the appendicular skele- centers are often presumed to be related inti- ton possesses several novel features whose mately to a mechanism of growth regulation presence cannot be attributed simply to trun- that confers a pattern of “determinate”growth cated development. Mesopodial elements, to those amniotes that possess them (Haines, which typically are cartilaginous throughout ’38, ’41, ’42, ’69; Romer, ’56, ’70).Tetrapods that life in plethodontid salamanders, are ossified in lack secondary centers are considered to have adult Thorius. Furthermore, long bones, which “indeterminate” growth (Goin and Goin, ’71; retain relatively large and well-developedcarti- Goss, ‘74, ’80; Romer, ’66). laginous epiphyses in adults of most other The dichotomy between determinate and in- plethodontid genera, show increased ossifica- determinate growth is well entrenched in the tion in Thorius. Yet despite the identification literature on skeletal development and has of these “derived features of the appendicular played a central role in arguments concerning skeleton more than a century ago (Cope, 1869a), the origin of particular vertebrate groups, such their role in limb function and skeletal matura- as lizards (Carroll, ’77).In many instances, the tion has remained unclear. In this paper, I pre- presence or absence of secondary ossification sent the results of an examination of skeletal centers is used as a sufficient criterion for iden- growth and variation in Thorius that was con- tifying which mode of growth prevails in a par- ducted as part of a larger study of the mecha- ticular taxon (for example, see Andrew and nisms and morphological consequences of ex- Hickman, ’74). Yet thorough studies of pat- treme size reduction in salamanders (Hanken, terns of skeletal growth on which the dichot- ’80). This allows consideration of both the omy is based include surprisingly few taxa; mechanism by which size reduction was furthermore, empirical data gathered from lon- achieved during the evolution of Thorius from gitudinal studies of growth of many natural larger ancestors and the means by which size populations of fish and amphibians suggest differentiation is effected among species with- that growth patterns in these groups may be in the genus. quite “determinate,” regardless of the absence Analysis of size and skeletal growth regula- of secondary ossification centers. Thus, much tion was based primarily on observations of remains to be learned concerning the mecha- five species that collectively span the range of nisms of growth and size regulation in verte- adult body size found in Thorius. Supplemen- SKELETAL GROWTH AND VARIATION IN SALAMANDERS 59 tary observations of seven additional species Abbreviations provided estimates of the range of skeletal var- b, compact bone fibula iation in the genus. I focused particularly on C, centrale in termedium the appendicular skeleton for two reasons: 1) cl, centrale 1 hypertrophied cartilage d, diaphysis marrow cavity the presence of several unique, or derived, fea- dl-2, distal carpal 1-2 mature cartilage tures not seen in larger plethodontid salaman- d3, distal carpal 3 carpus metatarsal 3 ders, with the presumption that the occurrence d4, distal carpal 4 proliferating cartilage of these features is related to the process of dl-2, distal tarsal 1-2 radiale d3. distal tarsal 3 tarsus resting cartilage size and growth regulation; and 2) the promi- d4-5, distal tarsal 4-5 tibiale nent role attributed to appendicular skeletal e, epiphysis tibia growth processes in the mechanism of overall f. fibulare ulnare growth regulation in most amniotes (seeabove). fe. femur ulna Specifically, I examined the following aspects of limb skeletal morphology and development: 1) variation in the appearance and number of mesopodial elements, both within and between species; 2) ossification patterns of mesopodial elements and long bones, including compari- sons of species differing in adult body size; and 3) histological modifications of skeletal tissues C during ontogeny, and the implications of these modifications for adult skeletal growth and maturation. D MATERIALS AND METHODS ofThorius E To date, nine species of Thorius have been formally described (Wake and Lynch, '76). Re- cently, I completed a taxonomic survey of the genus based on both electrophoretic and mor- phological analyses in which I identified the Fig. 1. Snout-vent length (SVL) of adult males (below) following seven additional (undescribed) spe- and females (above)of T. pennatulus (A); T. macdougalli IB); T. minutissimus (C): T. schmidti (D); and T. narisovalis (E). cies: T. sp. A (Puerto del Aire, Veracruz), T. sp. Mean, vertical bar; range, horizontal bar; 95% confidence B (Volcan Orizaba and Las Vigas, Veracruz), T. interval, rectangle. Sample size is in parentheses. sp. C (Cerro Pelon, Oaxaca), T. sp. D (Cerro Pelon, Oaxaca), T. sp. E (Cerro Pelon, Oaxaca), T. sp. F (Guerrero and western Oaxaca), and T. TABLE 1. Mean snout-uent length of adult males (M)and sp. G (northern Oaxaca) (Hanken, '80). Formal females (F) of five species of Thorius' descriptions will be presented elsewhere. ______Species Sex SVL SE N These species will be referred to below by letter T. pennatulus M 18.9 0.3 10 designation. F 18.9 0.3 10 T. macdougalli M 19.5 0.7 10 Osteological analysis F 22.3 0.7 10 The bulk of the osteological analysis was T. minutissimus M 22.6 0.3 9 F 24.1 0.4 9 based on observations of ontogenetic series of T. schmidti M 23.5 0.2 8 approximately 30 specimens (10 adult males, F 25.0 0.4 10 10 adult females, and 10 juveniles, when avail- T. narisovalis M 25.4 0.5 10 able) of each of the following five species: T. F 27.8 0.4 10 pennatulus, T. macdougalli, T. minutissimus, 'Measurements are in millimeters; SE. standard error; N. sample size. T. schmidti, and T. narisoualis, (Fig. 1, Table 1).Samples of approximately 20 specimens (10 adult males and 10 adult females) of each of tified by the presence of enlarged testes andlor seven additional species were used to identify convoluted vasa deferentia; adult females were mesopodial fusion combinations that are vari- identified by the presence of maturing follicles ably present in the genus: T. troglodytes, T. or swollen andlor convoluted oviducts. De- pulmonaris, T.sp. B, T. sp. C, T. sp. 0,T. sp. F, tailed information about specimens examined and T. sp. G. Sexually mature males were iden- (e.g., localities) is available in Hanken ('80)and 60 J. HANKEN upon request. All specimens are deposited in tions accompanying increased mesopodial and the Museum of Vertebrate Zoology of The Uni- long bone ossification. Preservation, decalcifi- versity of California at Berkeley. cation, paraffin sectioning, and subsequent Specimens were preserved initially in 10% staining in hematoxylin and eosin followed neutral-buffered formalin. External measure- standard histological procedures (Humason, ments of body size (snout to posterior end '79). of vent: SVL) were taken from the formalin- preserved material. Following external mea- RESULTS surement, all specimens were differentially Appearance and number of mesopodial stained for bone and cartilage by an alizarin elements red-alcian blue procedure slightly modified Carpus. Primitively, the carpus of pletho- from that of Wassersug ('76) and Dingerkus dontid salamanders consists of eight distinct and Uhler ('77). Linear skeletal dimensions cartilages: three proximal elements (ulnare, in- were measured with a binocular microscope termedium, and radiale); two centrally located fitted with an ocular micrometer. centralia (centrale and centrale 1); and three All specimens were scored for the presence of distal elements (distal carpds 1-2, 3, and 4) each of 15 potential combinations of fused me- (Wake, '63, '66). This pattern never is seen in sopodial elements and 18 different overall car- Thorius. Instead, many of these cartilages fuse pal or tarsal arrangements that were identified in at least nine different combinations: 1) ul- in a preliminary survey of 12 species. In the nare and intermedium; 2) ulnare, intermedium, vast majority of specimens, adjacent mesopo- and centrale; 3) distal carpal 4 and centrale; 4) dial elements were clearly either fused or sepa- distal carpal 3, distal carpal 4, and centrale; 5) rate. However, in a few cases adjacent distal carpal 1-2, distal carpal 3, distal carpal 4, elements appeared fused yet retained a con- and centrale; 6) distal carpal 1-2 and centrale; spicuous suture between them. For the pur- 7) radiale and centrale 1; 8) distal carpals 1-2 poses of this study, these instances are scored and 3; 9) distal carpals 3 and 4. (See Fig. 2 and as fused. Right and left sides were scored Table 2.) separately. Few of these fusion combinations are com- Each mesopodial element was scored for the mon; most occur only at low frequencies but presence of ossification (an element was con- may be found in several species (Table 2). The sidered ossified if it showed any alizarin- ulnare and intermedium are fused in all speci- stained deposit). For each carpus and tarsus, mens of all species. The centrale usually is an ossification index then was calculated that fused with distal carpal 4, but combinations equaled the percentage of total elements ossi- between the centrale and either ulnare-inter- fied (i.e., the number of ossified elements, di- medium or distal carpal 1-2 appear in low fre- vided by the total number of elements, multi- quency in many species. The fused distal car- plied by 100). Finally, separately calculated pal 4-centrale rarely joins distal carpal 3; it values for right and left sides were averaged to was fused with both distal carpals 1-2 and 3 in yield one carpus and one tarsus ossification in- one immature specimen of T. schmidti. Fusion dex value for each . of the radiale and centrale 1 was seen in only A second type of ossification index, calcu- one specimen each of T. narisovalis and T. sp. lated separately for both the right femur and D; the latter specimen also demonstrated the the right third metatarsal, was used to repre- only instance of fusion of distal carpals 3 and 4. sent the degree of ossification of long bones. The frequency of carpal fusion combination For each element, the length of the ossified di- 8-distal carpals 1-2 and 3-varies greatly aphysis (shaft) was expressed as a percentage among species. For example, although this of the total length of the element (diaphysis combination was seen in only 5-1070of the car- plus both cartilaginous epiphyses). The border pi of T. narisoualis, it characterized approxi- between adjacent regions of cartilage and bone mately half of the T. pennatulus examined. usually was well defined. However, in large fe- Variable occurrence of these nine fusion com- murs showing the maximal amount of ossifica- binations, both within and among species, tion, epiphyseal cartilage at each end was re- yields nine different overall carpal arrange- duced to a thin cap, or shell, surrounding a ments in which the actual number of separate bony core. This maximum condition was opera- carpal units ranges from four to seven (Fig. 2). tionally defined as 99% ossified. As many as four different arrangements may One hindlimb from both juvenile (SVL = be present in a single sample of 20 or fewer con- 12.5 mm) and adult (SVL = 30.9 mm) speci- specific individuals from a single locality (Ta- mens of T. sp. F were examined in 10-pm serial ble 3). The most common pattern in all species sections to identify the histological modifica- is I: ulnare-intermedium, radiale, centrale 1, SKELETAL GROWTH AND VARIATION IN SALAMANDERS 61

23 1

Primitive Plethodont id Pattern

Fig. 2. Alternate carpal arrangements in Thorius (dorsal ment to another. Cartilage is stippled, except in view). All arrangements are drawn as right carpi, although mesopodials, which have been left unshaded. Arrangements some were observed in the left carpus only (see below). were drawn from the following specimens (asterisk denotes Pattern I predominates in all species. The remaining left carpus): I) T narisoualis. M 4456; 11) T. narisoualis. M patterns are rare, except for pattern 11, which occurs in low 4518; 111) T. narisoualis, M 4527; IV) T. sp. F, M 6058; V) T. to moderate frequency in most species (Table 4). Arabic schmidti, M 3055; VI) T. narisoualis, M 4523; VIP)T. sp. D, numerals identify the carpal fusion combination(s) HBS 2203; VIII*) T. sp. G, M 3808; IX) T. troglodytes. M (described in text) involved in transition from one arrange- 5515. TABLE 2. Frequency (Yo) of nine carpal fusion combinations in adult Thorius‘,’

(2) (3) (4) (7) (8) Ulnare-intermedium Distal carpal 4 Distal carpal 3+ Radiale + Distal carpal 1-2 + centrale + centrale d4-centrale centrale 1 + distal carpal 3 Species Unfused Fused Unfused Fused Unfused Fused Unfused Fused Unfused Fused T. pennatulus 100 100 97.4 2.7 100 48.8 51.2 (N = 19.20)’ T. macdougalii 100 100 100 - 100 85.0 15.0 (N = 20) T. minutissirnus 100 100 91.7 8.4 100 83.4 16.7 (N = 18) T. schmidti 100 100 94.4 5.6 100 88.9 11.1 (N = 18) T. narisoualis 100 100 97.5 2.5 97.5 92.3 7.8 (N = 20.19)’ T. troglodytes 100 97.4 97.4 2.7 100 97.4 2.7 (N = 19) T. pulmonaris 100 100 100 - 100 91.4 8.6 (N = 11) T. sp. B 100 100 100 - 100 51.2 48.9 (N = 18,19)3 T. sp. C 100 100 97.5 2.5 100 90.0 10.0 (N = 20) T. sp. D 100 97.5 92.3 7.8 97.5 87.1 12.9 (N = 19,20)3 T. sp. F 97.1 97.1 100 - 100 83.0 17.0 (N = 17.18)3

T. sp. G 97.2 97.2 100 I 100 94.5 5.6 (N = 181

‘In each species, frequencies were first calculated separately for right and left sides and then averaged to yield a single value. ‘Combination 1, ulnare and intermedium. is found in all specimens Combination 5. distalcarpal 1-2, distalcarpal 3, anddistal carpal 4-centrale, was seen in only one juvenile T. schmidti (both carpi). Combinations 6. distal carpal 1-2 and centrale. and 9, distal carpal 3 and distal carpal 4, were seen each in only one of twenty specimens of T sp. D (left carpus). ’Sample sizes for the right and left sides, respectively. SKELETAL GROWTH AND VARIATION IN SALAMANDERS 63 distal carpal 4-centrale, distal carpal 1-2, and six potential fusion combinations were pres- distal carpal 3. Fusion of distal carpals 1-2 and ent, had five to eight separate tarsal units 3 produces a second pattern (11)that occurs in (Fig. 3). In contrast to the carpus, in which the low to moderate frequency in most species. same carpal arrangement predominates in Pattern 111, which differs from the most com- nearly all species (Table 3), each of four tarsal mon pattern in the fusion of distal carpal 3 arrangements predominate in at least one, and with distal carpal 4-centrale, is low in frequen- often several, species (Table 5). Pattern I dif- cy (510%)in at least seven species: T. minutis- fers from the primitive plethodontid arrange- simus, T. schmidti, T. pennatulus, T. narisova- ment only in the fusion of distal tarsals 4 and 5; lis, T. troglodytes, T. sp. C, and T. sp. D. The presumably it represents the primitive condi- remaining six patterns are rare; each occurs in tion in Thorius. It characterizes most T. minu- only one species. tissimus, T. macdougalli, T. narisovalis, T. Tarsus. The primitive arrangement of the troglodytes, T. sp. C, and T. sp. D, and many T. tarsus in plethodontid salamanders comprises schmidti. Pattern V has an additional fusion of nine separate cartilages: three proximal ele- the intermedium and fibulare; it appears in ments (fibulare, intermedium, and tibiale); two most T. pulmonaris and T. sp. G, and in many centralia (centrale and centrale 1);and four dis- T. schmidti, T. sp. B, and T. sp. F. Pattern VII tal elements (distal tarsals 1-2, 3, 4, and 5) has an additional fusion of the centrale and dis- (Wake, '63, '66). As in the carpus, fusions of tal tarsal 4-5, whereas pattern VIII has yet an these elements reduce the actual number of additional fusion of distal tarsals 1-2 and 3; to- separate tarsal units present in a given speci- gether these patterns characterize nearly all T. men of Thorius to five to eight. The following pennatulus. The remaining five tarsal arrange- six tarsal fusion combinations were observed ments (11-IV, VI, IX) may be derived from (Table 4): 1) intermedium and fibulare; 2) inter- these four patterns by additional fusions. Each medium and centrale; 3) distal tarsals 4 and 5; occurs infrequently in a given species, but the 4) centrale and distal tarsal 4-5; 5)centrale, dis- same pattern may appear in many different tal tarsal 3, and distal tarsal 4-5; 6) distal tar- species. As many as five different tarsal ar- sals 1-2 and 3 (Fig. 3). rangements may be present in a sample of 20 Distal tarsals 4 and 5 are always fused. They or fewer individuals from a single locality (e.g., fuse with the centrale in all T.pennatulus, but T. minutissimus; Table 5). this combination appears in only low to moder- Finally, variation in mesopodial number in- ate frequency, if at all, in other species. Distal cludes a high frequency of right-left asymme- tarsal 3 is fused with distal tarsal 1-2 at low to try in mesopodial fusion and, therefore, overall moderate frequencies in many species, espe- carpal or tarsal arrangement (Table 6). For ex- cially T. macdougalli. The typically separate ample, approximately one-fourth of the sample intermedium and centrale are fused only in the of each of several species were asymmetric for right tarsus of one of 17 specimens of T. carpal fusion combination (8), distal carpal 1-2, minutissimus. and distal carpal 3. Combining the data con- Fusion of the intermedium and fibulare ex- cerning all 11 different mesopodial fusion com- hibits the most striking pattern of variation. binations observed in the five species of Thor Here, species may be arranged in a cline based ius examined in detail, between 20% and 70% on the frequency of intermedium-fibulare fu- of the specimens of a given sample were asym- sion, from species in which the elements are metric for at least one mesopodial fusion com- nearly always separate (T. minutissimus, T. bination and, therefore, carpal or tarsal narisovalis), to species of intermediate fre- arrangement. quency of fusion (T. macdougalli, T. sp. F), to species in which the two elements are always Ossificationpattern or nearly always fused (T. schmidti, T. penna- Mesopodial elements tulus). Fusion frequency bears no obvious rela- General observations concerning the timing tionship to body size; two species of similar of mesopodial ossification apply equally to the adult size may exhibit greatly different fusion carpus and tarsus. Typically, mesopodial ele- frequencies (e.g., T. pennatulus and T. mac- ments are cartilaginous in both juvenile and dougalli), whereas a given fusion frequency adult plethodontid salamanders (Wake, '66). may be shared by two species differing in mean Although the elements are cartilaginous in ju- adult size (e.g., T. narisovalis and T. venile Thorius (Fig. 4A), nearly all adults have minutissimus). some or all mesopodial elements ossified (Fig. I observed nine different overall tarsal ar- 4B,C). In each species, intermediate values of rangements that, depending on which of the carpal or tarsal ossification index occur in 64 J. HANKEN

TABLE 3. Frequency (%) of nine alternate carpal arrangements in adult Thorius’,’ Species I I1 Ill IV-1x3 T pennatulus 46.2 51.2 2.7 -

IN = 19.201‘.I T macdougalli 85.0 15.0 - (N = 20) T. minutissirnus 75.0 16.7 8.4 (N = 18) T. schmidti 83.3 11.1 5.6 - (N = 18) I: narisovalis 87.3 7.8 2.5 2.5 (VI) (N = 20,19)+ T troglodytes 92.1 2.7 2.7 2.7 (1x1 (N = 19) I: pulmanaris 91.0 9.1 (N = 11) T sp. B 51.2 48.9 - (N = 19,18)‘ T. sp. c 87.5 10.0 2.5 (N = 20) I: sp. D 76.9 12.9 7.8 2.5 (VII) = 19.20P IN 1. T. sp. F 80.1 17.0 3.0 (IV) (N = 17.18)’ T sp. G 94.5 2.8 2.8 (VIII) (N= 18) ‘Ineach species, frequencies were calculated separately for the right and left sides and averaged to yield a single value. ’I: u-i. r. cl. dl-2, d3. d4-c; 11: u-i. r, el, (dl-Z)-d3,d4-c; 111: u-i. r, cl. dl-2. d3-(d4-cj; IV: u-i-c, r, cl. dl-2,d3, dQ;V. u-i, r. cl. (dl-Z)-d3-d4-c; VI: u-i, r-cl, dl-2, d3, d4-c; VII: u-i, r-cl, (dl-2)-c, d3-d4; VIII: u-i-c. r. c1, Idl-Z)-d3, d4; IX u-i, r, c, ci, dl-2, d3, d4. >PatternV was seen in only a single, juvenile specimen of ‘I: schrnidti (both carpi). ‘Sample sizes for the right and left sides, respectively.

TABLE 4. Freouencv 1%) ofsix tarsal fusion combinations in adult Thorius‘

(1) (4) (6) Intermedium t Centrale + Distal tarsal 2 + fibulare .__ distal tarsal 4-5 ~- distal tarsal 3 Species Unfused Fused Unfused Fused Unfused Fused ._ ___~ T pennatulus - 100 - 100 56.6 43.4 (N = 20,19)’ T. macdougalli 73.7 26.3 97.4 2.7 94.7 5.3 (N = 19) I: minutissimus 94.3 5.8 97.2 2.8 97.2 2.8 (N = 17,18)’ T. schmidti 11.1 88.9 63.9 36.1 97.2 2.8 (N = 18) I: narisoualis 90.0 10.0 95.0 5.0 95.0 5.0 (N = 20) T. troglodytes 92.4 7.7 100 - 100 (N = 20,19)3 T. pulmonaris 18.2 81.8 100 100 (N = 11) T sp. B 52.5 47.5 100 71.5 28.5 (N = 17,19)3 T. sp. c 100 100 97.2 2.8 IN = 18.16)”.. T. sp. D 94.3 5.8 100 - 88.6 11.5 (N = 18,17)3 T sp. F 48.7 51.4 97.2 2.8 100 - (N = 18,17)’ T. sp. G 5.6 94.4 97.2 2.8 100 - (N = 18)

‘In each species, frequencies were calculated separately for the right and left sides and averaged to yield a single value. ‘Combination 3, distal tarsals 4. and 5. was found in all specimens. Combinations 2. intermedium and centrale. and 5. centrale. distal tarsal 3, and distal tarsal 4-5, were seen only in single specimens (right tarsus) of 1: minutissirnus and T. sp. E: respectively. ’Sample sizes for the right and left sides, respectively. l6 )------I 1. I mm

Pr im it ive Plethodontid Pattern -6

Fig. 3. Alternate tarsal arrangements in Thorius (dorsal from one arrangement to another. Cartilage is stippled, view). All arrangements are drawn as right tarsi, although except in mesopodials, which have been left unshaded. some were observed in the left tarsus only (see below). Four Arrangements were drawn from the following specimens patterns (I, V. VII, and VIII) each predominate in at least (asterisk denotes left tarsus): I, III*) E nansoualis, M 4518: one species. The remaining patterns occur only in low 11) E ~n'soualis,M 4456: IV) I: minutissimus, M 4583; V) frequencies, but each may be present in several species E nanwualis, M 3471; VIJ E sp. F, MVZ 110669: VII) 'I: sp. (Table 4). Arabic numerals identify the tarsal fusion F, MVZ 110644; VIII) 'I: schmidti, M 5173; IX) T. sp. F, M combination(s)(described in text) involved in the transition 6069. 66 J. HANKEN

TABLE 5. Frequency (%J of nine alternate tarsal arrangements in adult Thoriu~'.'.~ Species I I1 Ill V VI VII VIII T. pennatulus (N = 19.20) I: macdougalli 71.1 23.7 (N = 20) T minutissimus 85.8 5.8 (N = 18) T. schmidti 11.1 52.8 (N = 18) T. narisovalis 80.0 10.0 (N = 20.19Y T. troglodytes 92.4 7.7 (N = 19) 1: pulmonaris 18.2 - 81.8 IN,~ = -11) . T sp. B 38.1 14.4 33.5 (N = 19.18Y I: sp. c 97.1 2.8 (N = 20) T. sp. D 82.9 11.8 5.8 IN = 19.20)' T. sp. F 48.7 - - 45.8 - 2.8 - (N = 17,18)4 I: sp. G 5.6 - - 91.7 - 2.9 - (N = 18)9

'In each species. frequencies were calculated separately for the right and left sides and averaged to yield a single value. 'I: i. f. t, c. cl. dl-2. d3. d4-5: 11: i, f, t, el, c-(d4-5),dl-2, d3: 111: i, f. t, c. cl. (dl-2)-d3, d4-5; IV i-c. f, t. cl, dl-2. d3, d4-5; V: i-f. t, c, cl. dl-2,d3, d4-5; VI: i-f, t, c, el, (dl-Z)-d3, d4-5: VII: i-f, t. cl, c-(d4-5). dl-2. d3; VIII: i-f. t. cl, c-(d4-5), ldl-%d3: IX i-f, t, cl, c-d3-(d4-5). 'Patterns IV and IX were found only in single specimens (right side) of T. minutissimus and T. sp. F, respectively. 'Sample sizes for the right and left sides, respectively.

TABLE 6. Frequency (%)of right-left asymmetry of carpal fusion combination @-distal carpal 1-2 and distal carpal 3- and overall card and tarsal arrawement Overall carpal and Carpal fusion (8) tarsal arrangements Svmmetric Asvmmetric Svmmetric Asymmetric I: pennatulus 14 5 6 13 (N = 19) (73.7) (26.3) (31.6) (68.4) T. macdougalli 16 4 14 6 (N = 20) (80.0) (20.0) (70.0) (30.0) T. minutissimus 13 5 11 7 (N = 18) (72.2) (27.8) (61.1) (38.9) 'I: schmidti 14 4 6 12 (N = 18) (77.8) (22.2) (33.3) (66.7) T. narisoualis 18 1 15 4 IN = 19) 194.71 I 5.31 178.9) 121.11

small mature specimens which collectively oc- usually is the first carpal unit to ossify. How- cupy a relatively narrow range of body size ever, there is frequent variation among speci- (Fig. 5A-E). This indicates an abrupt onset mens with respect to both carpal and tarsal os- and completion of mesopodial ossification co- sification sequences; some elements tend to incident with sexual maturation. ossify earlier than others, but exceptions are Ossification sequences of carpal and tarsal common. Below, I describe only the most typi- units were determined by examination of onto- cal patterns. There were no apparent differ- genetic series of small adults. For example, in ences in ossification sequence among species. those specimens with only one ossified carpal, Carpus. The carpus usually ossifies from this unit typically was the fused ulnare-inter- seven separate centers. The sole exception medium. Thus, the fused ulnare-intermedium noted was the fused distal carpal 4 and cen- SKELETAL GROWTH AND VARIATION IN SALAMANDERS 67

Fig. 4. Cleared and differentially stained (bone and carti- and C. Tarsals are cartilaginous in A but the intermedium lage) right hindlimbs of juvenile and adult T. narisoualis and fibulare are partly ossified (appearing black in (dorsal view) showing ossification of tarsal elements and photograph) in B; all the tarsals are at least partly ossified long bones. A. M 4443, juvenile male, SVL = 21.8 mm; B. M in C. Note the fused intermedium - fibulare in A, the fused 4453, adult female, SVL = 26.8 mm; C. M 4456, adult distal tarsal 4-5 and centrale in C, and the reduced phalan- female, SVL = 28.4 mm. The cartilaginous epiphyses geal number of the second digit in C. Bar equals 0.5 mm. present in the long bones of A are reduced or lacking in B

trale, which, though usually ossifying as a sin- were numerous exceptions. For example, the gle center, ossified from two separate centers tibiale, fibulare, centrale 1, and distal tarsal 4-5 in one carpus of one adult specimen of T. sp. F each were ossified before.the intermedium in at (SVL = 27.1 mm), making a total of eight cen- least one specimen. ters in this carpus. The predominant ossifica- The five Thorius species differ in mean adult tion sequence is: ulnare-intermedium (ossify- body size (Table 1; Fig. 1).The body size at ing as two centers); distal carpal 4-centrale; which mesopodial ossification is initiated also radiale; centrale 1; distal carpal 1-2 or distal differs between species, corresponding to a carpal 3. This sequence differs slightly from shift in the timing of sexual maturity relative that of Wake (’66)who, citing the observations to body size among species. For example, in T. of Uzzell(’61),reported that the radiale ossified pennatulus, the smallest species, sexual ma- before distal carpal 4-centrale. turity and carpal ossification commence at Tarsus. Eight separate centers ossify in body sizes as small as 15 mm SVL, and carpal the tarsus in the following sequence: interme- ossification is complete by 20 mm SVL (Fig. dium; fibulare; centrale, tibiale, centrale 1 or 5A). Thorius macdougalli, with a larger mean distal tarsal 4-5; distal tarsal 1-2; distal tarsal adult body size, typically initiates sexual ma- 3. This sequence, too, represents only some of turity and carpal ossification at body sizes the ossification sequences examined as there greater than 20 mm SVL, although carpal ossi- 68 J. HANKEN . A 6 ..A ‘O0175 A .A A

25 0

75 .u AA A .A 25 .

MF 100- .I.IUA E . A A 75 - . A 50- . 25-

0 - ((OD wmAA .....1 . I

Fig. 5. Carpal mineralization index vs. body size in T adult female, ( 0)juvenile. Mean adult size of adult males pennutulus (A);T. mucdougulli (B);T rninutissimus (C); 7: (M) and females (F) of each species is indicated on the schrnidti (D); and T. nQlisOUUliS (E). (a)adult male, (A) abscissa. SKELETAL GROWTH AND VARIATION IN SALAMANDERS 69 fication may be complete in less than gradually, beginning well before sexual matur- 25 mm SVL (Fig. 4C). And in T. narisoualis, the ity. Finally, species differ in the degree of meta- largest species, maturity and ossification are tarsal ossification over a broad range of body delayed until nearly 25 mm SVL, and ossifica- size, again in a regular manner correlated with tion may be incomplete in animals as large as species differences in mean adult body size and 30 mm SVL or more (Fig. 5E). size at sexual maturity. Femur Histological modifications during ontogeny Epiphyses of small juvenile specimens occu- Examination of serial sections of hindlimbs py as much as 30% of femur length and may in- of juvenile and adult Thorius allowed determi- clude even the proximal and distal ends of the nation of the exact nature of mesopodial and central shaft (Fig. 4A). With increasing size, long bone mineralization, and characterization the proportion of femur length ossified in- of important differences in growth parameters creases gradually until in the largest speci- of the appendicular skeleton before and after mens each epiphysis is reduced to a thin carti- sexual maturity. In describing the histology of laginous cap, or shell, surrounding a bony core these mineralizing tissues I employ the termi- (Fig. 4B,C). nology of Andrew and Hickman (’74). Plots of femur ossification index versus Mesopodials of juvenile specimens are en- body size for each species are presented in Fig- tirely cartilaginous, with neither internal cavi- ure 6A-E. As was observed in the ossification ties nor mineralization (Fig. 8A). Long bones, of mesopodial elements (Fig. 5A-E), relative such as the femur, of juveniles have well-devel- ossification of the femur is a function of body oped cartilaginous epiphyses that fill the entire size. However, unlike the abrupt “steplike”ap- volume of each bulbous end and adjacent re- pearance of carpal ossification that occurs over gions of the narrow shaft (Fig. 9A). A layer, or a relatively narrow range of body size in each zone, of resting cartilage forms the articulat- species coincident with sexual maturation, os- ing facet peripherally and extends centrally to- sification of the femur is a gradual process that wards the shaft for approximately half the begins well before the onset of sexual matur- length of the epiphysis. There it grades into a ity. Femurs of most adults of all species are ful- layer several cells thick comprising columns of ly (i.e., 99%) ossified, but femur ossification flattened, compressed, dividing cells - the so- differs greatly among juveniles, with degree of called zone of proliferating cartilage- that ossification proportional to body size within gives way to a zone of hypertrophied, or matur- each species. ing, cartilage near the base of the epiphysis. In The pattern of increased ossification with in- the neck of the shaft a final, thin, poorly de- creased body size is evident in each species. fined layer of mature cartilage cells is dis- However, species differ in the degree of femur rupted and replaced by a marrow cavity that ossification over a broad range of body size. fills the interior of the surrounding bony diaph- For instance, in T. pennatulus femur ossifica- ysis. This complex of stratified layers of skele- tion is complete by 20 mm SVL, whereas in T. togenous tissue in successive stages of differ- narisoualis, a much larger species, complete fe- entiation and maturation, especially the mur ossification typically is attained only at proliferative zone, is characteristic of actively body sizes in excess of 25 mm SVL; juvenile T, growing bone (Andrew and Hickman, ’74;Ham narisoualis of a body size comparable to that of and Cormack, ’79). adult T. pennatulus have no more than 80% of The appearance of adult mesopodials is strik- the femur ossified (cf. Fig. 6A,E). Here, too, as ingly different from that of juvenile elements was seen in the ossification of mesopodial ele- (Fig. 8B). Cartilage, which fills the mesopodial ments (see above),differences among species in elements of juveniles, is reduced to a thin layer the degree of femur ossification at a given body of resting cartilage surrounding a large inter- size parallel species differences in mean adult nal marrow cavity in adults. This layer is thick- body size at sexual maturity. est where it forms articulating facets with ad- jacent elements, but it is never more than two Third metatarsal to four cells deep and there are few dividing Ossification of the third metatarsal demon- cells. Peripherally, a thin layer of compact strates the same general phenomenon as was bone surrounds this cartilaginous layer, except observed for the femur. Ossification is least in at the articulating facets. These histological small specimens, in which cartilage may con- modifications of adult mesopodials are indica- stitute up to 40% of the length of the element, tive of intracartilaginous, or endochondral, os- and is proportional to body size within each sification, and not calcified cartilage as has species (Figs. 4, 7A-E). Ossification increases been claimed previously by Uzzell(‘61). 70 J. HANKEN 100

a a 0. 90 A 0 0 aA 8: 08 8C

70 1 I I I 10 f20 30 10 f+ 30 M,F MF 00- C 7 ImllIlH D pP AA YA aA A 0 A 90-, - A -A 0 - **.. 80-* ;. 0 00 0. 0. 0 70 I I I I I I 1

100 - mamiiwa A E *NII A A 90-

0. ..a ..a 80- 0 0:.

0

70 I I

SVL (mm)

Fig. 6. Femur ossification index vs. body size in T. adult female, ( 0)juvenile. Mean adult size of males (M)and pennatulus (A);Z rnacdougalli (B);T. minutissirnus (C);T. females (F)of each species is indicated on the abscissa. schmidti (D); and T. narisoualis (E). (a)adult male, (A) SKELETAL GROWTH AND VARIATION IN SALAMANDERS 71

100. A B

A 90 8 =s 80 A 0: 0 0 70 0 n 60 Us 50 I I I I 1 M +' 30 4 +' 30 - M.F MF 0 too C 0 f", 0 t 90 0 t Q) E 80

Y- O 70*

c 60 .-0 4- 0 .-0 50 I 1 'c 1 2b t ;o 1 2'0 vt 30 u) MF MF u) 0 E loo 1

601

50 f I i 15 I+ t I 35 MF

Fig. 7. Metatarsal 3 ossification index vs. body size for adult female, ( 0)juvenile. Mean adult size of males (M)and T.pennatulus (A);T. macdougalli (B);T. minutissimus (CI; T. females (F)of each species is indicated on the abscissa. schmidti (D); and T. narisoualis (E). (M) adult male, (A) 72 J. HANKEN

Fig. 8. Hematoxylin and eosin-stained sections through mm). B. Ulnare and adjacent distal portion of ulna of an carpal elements of juvenile and adult Thorius. A. Ulnare, adult female specimen of T sp. F (TJP 14569; SVL = 30.9 intermedium, and adjacent distal portion of ulna of a mm). juvenile specimen of T minutissimus (M 4636; SVL = 12.5

Correlated with mesopodial ossification is skeleton has lost the physiological capacity for extensive reduction of the cartilaginous epi- longitudinal growth and elongation that is typ- physis in adult long bones (Fig. 9B). In each ical of juvenile elements. epiphysis, cartilage is limited to a peripheral layer of articulating cartilage one to three cells DISCUSSION thick that sheathes the joint articulation. Skeletal growth pattern in Thorius Zones of proliferating and hypertrophied carti- The appendicular skeleton of Thorius, as lage, both of which are conspicuous in juve- compared to that of most plethodontid sala- niles, are absent, although a few cells in the ar- manders, demonstrates conspicuous and ex- ticulating layer are dividing. The marrow cavity tensive increase in the degree of ossification and its surrounding layer of bone - which in j u- during ontogeny. Increased ossification of veniles are restricted to the narrow shaft and long bones occurs as a gradual process, begin- barely reach the base of each expanded end- ning prior to sexual maturity, and is propor- have invaded both ends and filled their entire tional to body size in each species; maximum volume below the thin layer of epiphyseal carti- ossification is achieved soon after sexual matu- lage. The extreme reduction of cartilage, espe- rity (Fig. 6). However, unlike bone growth in cially the proliferative layer, and the conse- most amniotes, Thorius lacks secondary ossifi- quent nearly complete ossification of both cation centers at all stages. Instead, increased ends of the femur and adjacent mesopodial ele- ossification is solely the result of longitudinal ments, suggest that the adult appendicular expansion of the primary (diaphyseal)center of SKELETAL GROWTH AND VARIATION IN SALAMANDERS 73

Fig. 9. Longitudinal sections through femurs of juvenile Distal end of femur of an adult T sp. F (TJP 14596; SVL = and adult Thon‘us. A. Proximal end of femur of a juvenile 30.9 mm). specimen of T minutissirnus (M 4736: SVL = 12.5 mm). B ossification. In contrast, mesopodial ossifica- appendicular skeleton contrasts sharply with tion appears abruptly and coincidentally with the otherwise “reduced,” pedomorphic mor- sexual maturity (Fig. 5); it may be seen as phology that generally characterizes the skele- merely the culmination of a general maturation ton of Thorius, particularly the limbs and skull process that involves the entire limb skeleton. (Wake, ’66). In view of the relation of the in- Increased ossification involves additional creased ossification to the pattern of determi- qualitative, as well as quantitative, changes. nate appendicular skeletal growth, it seems Most important among these is the loss of the likely that these novel features are only part of complex of stratified layers of skeletogenous a more fundamentai mechanism of overall tissue from the ends of long bones, especially growth regulation that restricts adult body the proliferative zone, and the consequent re- size in Thorius by precocious truncation of so- duction in the size and development of cartilag- matic development relative to that observed in inous epiphyses (Fig. 9). As a result, and as is larger, generalized plethodontid salamanders. the case in most amniotes, the physiological The novel mineralizations in the appendicu- capacity for longitudinal growth of the appen- lar skeleton of Thorius were first identified by dicular skeleton in juveniles is absent from E.D. Cope more than a century ago in his origi- adults. This constitutes a mechanism of deter- nal description of the genus (1869a).Cope pre- minate growth unique to urodele amphibians sented the mineralizations as a taxonomic and analogous to that found in most amniotes. character that distinguished Thorius from all The extensive ossification of much of the adult other plethodontid genera, and most subse- 74 J. HANKEN quent discussions of the mineralizations have Thorius. If these factors do influence ossifica- considered them only in this context (for exam- tion events in the limb, it is as a result of an ple, see Cope, 1869b; Dunn, ’26; Hall, ’52; Hil- interaction between these stimuli and a predis- ton, ’46, ’48; Rabb, ’55; Taylor, ’44).Two excep- position of these cartilages to increased ossifi- tions are the works of Uzzell (’61) and Wake cation conferred by physiological alterations (’66),who offered functional (i.e., biomechani- associated with sexual maturation. cal) models to explain the increased mineraliza- Increased ossification of the appendicular tion in the appendicular skeleton. Uzzell also skeleton in Thorius is a derived condition with described variation in mesopodial ossification respect to larger, generalized plethodontid sal- in a single species, T. pulmonaris. amanders that display the primitive condition Wake proposed that the increased ossifica- of well-developed cartilaginous epiphyses and tion of long bones acted as “strengthening cartilaginous mesopodial elements in both ju- compensations for paedomorphic weakening” veniles and adults (Wake,’66). However, ossifi- (66: 46), an explanation similar to that claimed cation of mesopodial elements actually repre- earlier by Uzzell for the ossified mesopodial sents the secondary appearance of a feature elements. Both authors cited the independent that primitively was present in urodeles but development of one or both of these traits in was lost by ancestors of modern plethodontid other genera of small salamanders as additional salamanders (Romer, ’66). Thus, the presence evidence in support of their ideas (see Rabb, of ossified mesopodials may be interpreted in ’55). However, while mechanical influences can at least two ways. In one, mesopodial ossifica- have a dominant role in effecting both the se- tions represent an atavism - the reappearance quence and extent of skeletal development in of bones homologous to those of nonplethodon- vertebrates generally (Abdalla, ’79; Hall, ’71, tid salamanders as a result of the reactivation ’75, ’78; Hoyte and Enlow, ’66; Murray, ’36; of a developmental “program”that is otherwise Murray and Drachman, ’69) and have been in- dormant in plethodontid salamanders (see Rai- voked to explain the calcification or ossifica- kow (’75)for an example from birds)-whereas tion of hyoid cartilages in many salamanders, in the other, ossified mesopodials are a neo- including Thorius (Lombard and Wake, ’77; morph, a truly novel morphological feature Ozeti and Wake, ’69;Uzzell, ’62),I consider bio- with a different developmental basis from mechanical explanations of the unique limb those seen in nonplethodontid salamanders. mineralizations of Thorius unlikely for several Because mesopodial ossification appears regu- reasons. larly as part of a coordinated mechanism of ap- First, Uzzell (’61) inferred that force trans- pendicular skeletal growth regulation that is mission was greatest through postaxial meso- unique to Thon’us, and not as an independent podia1 elements because they were the first to or rare developmental anomaly, I favor the lat- mineralize. Yet, as indicated by Wake (’66),the ter hypothesis. postaxial elements are lost or reduced most fre- quently in plethodontid salamanders, and thus Interspecific size differentiation, skeletal they seem to play a minor role in force trans- variation, and morphological novelty mission through the carpus and tarsus; they While the skeletal growth pattern of Thorius would not be expected to mineralize first if differs greatly from that of other plethodontid mineralization occurred in response to mechan- salamanders, it is strikingly similar in all spe- ical stress. Second, my observations of the cies of Thorius examined so far. In fact, the on- variability of the sequence of mesopodial ossi- ly major difference among species with respect fication suggest that the sequence cannot re- to limb ossification phenomena involves varia- flect the pattern of force transmission unless tion in the timing of these processes relative to the pattern itself is highly variable. Most sig- body size -species characterized by a relative- nificantly, if ossification occurred in response ly small mean adult body size initiate and com- to mechanical stress we would expect ossifica- plete ontogenetic increase in ossification at a tion to appear at a more or less constant size in smaller body size than do larger species (Figs. all animals. Instead, interspecific comparisons 5-7). Thus it appears that alteration in the tim- have revealed the onset of ossification to be ing, relative to body size, of processes confer- correlated with the onset of sexual maturation, ring determinate skeletal growth has been rather than a single body size for the genus. employed as a means of achieving adult size Therefore, it is unlikely that mechanical fac- differentiation within the genus. This provides tors play a primary role in effecting increased further evidence of the dominant role of heter- ossification in the appendicular skeleton of ochrony (sensu Gould, ’77)in effecting morpho- SKELETAL GROWTH AND VARIATION IN SALAMANDERS 75 logical diversification among plethodontid ple, Larson et al. ('81)identified the appearance salamanders (Alberch, '80, '81; Alberch and Al- of novel carpal and tarsal arrangements as one berch, '81; Larson, '79; Wake, '66). of two "key innovations" that permitted the The regularity and precise timing of limb os- adoption of an arboreal existence by the ances- sification phenomena in Thorius are superim- tors of the genus Aneides. They al- posed on a pattern of extreme variation in the so cited the absence of intermediate (i.e., tran- actual composition of the limb skeleton, as for sitional) arrangements as evidence that the example with the variation in mesopodial fu- unique arrangements in Aneides arose in "sin- sion combinations and overall carpal and tar- gle steps" from the presumed ancestral ar- sal arrangement (Tables 2-5). Both the carpus rangements seen in the closely related genus and tarsus manifest trends toward reduction Plethodon. In Thorius, certain carpal and tar- in the number of separate elements, as cartilag- sal arrangements do distinguish some species. inous units that are separate in the respective But several other arrangements, which are as presumed ancestral arrangements coalesce to different as those that distinguish species of give as few as four elements in the carpus (Fig. Thorius from each other (and as different as 2) and five units in the tarsus (Fig. 3). More- those that distinguish Aneides from Pletho- over, decrease in mesopodial number has don),are present together within restricted lo- evolved independently several times, as re- cal populations of each of many species of vealed by the different carpal or tarsal arrange- Thorius. In some populations, mesopodial ments that contain the identical (reduced) polymorphism involves as many as four differ- number of separate elements (e.g., carpal pat- ent carpal or five different tarsal arrangements terns 11, 111, VI, VIII). Six carpal arrange- and may include a high frequency of right-left ments (111-VIII) and at least four tarsal ar- asymmetry in single specimens. In addition, rangements (11-IV, VI) are not found in other stages intermediate between fused and un- plethodontid salamanders (Wake, '66); thus fused states were observed occasionally in miniaturization of the appendicular skeleton in many combinations, as has been observed in Thorius has been accompanied by the appear- detailed analyses of the development and gene- ance of novel morphological arrangements not tic basis of mesopodial fusion in other verte- found in ancestors. However, variation in the brates (Griineberg, '63; Griineberg and Hus- number of limb elements is only part of a gen- ton, '65; Wright, '34). In Thorius, the eral pattern of extreme skeletal variation asso- establishment of novel morphological features ciated with the trend towards skeletal reduc- of the limb skeleton (often comparable in mag- tion in the genus (Wake, '66). Interestingly, a nitude to those distinguishing species, or even similar pattern of extreme variation and novel- genera) may require no mechanism other than ty in the composition of limb skeletal elements the fixation of a particular trait that occurs accompanying decreased relative limb size is variably within a single population. common in many vertebrate lineages, such as the lizard genus Bachia (Lande, '78) and the Skeletal growth regulation in tetrapods plethodontid salamander genus Oedipina Mammals and birds demonstrate a pattern (Wake, '66), suggesting that increased skeletal of determinate growth in which maximum variation, and the appearance of novel morpho- adult body size is attained relatively early in logical arrangements, may represent an inevi- life, and it is clear that many skeletal features table "by-product" of reduction via develop- peculiar to these groups, particularly the pres- mental truncation (see also Marshall and ence of secondary (epiphyseal)ossification cen- Corruccini, '78). ters, are related to this mechanism of overall The magnitude of carpal and tarsal variation growth regulation. However, many authors in Thon'us is exceptional among amphibians. have used the association between secondary Intraspecific and, more importantly, intrapop- ossification centers and determinate skeletal ulational variation in carpal and tarsal growth in these groups to claim the absence of arrangement is rare in most plethodontid sala- determinate skeletal growth in tetrapods that manders, although mesopodial fusion combi- lack secondary centers (e.g., Andrew and Hick- nations distinguish many plethodontid genera man, '74; Carroll, '77; Goss, '74, '80;Haines, '38, and, in some cases, individual species from '42, '69). This claim is unjustified for at least congeners (Alberch, '80, '81; Larson et al., '81; two reasons. First, fusion of primary and sec- Wake, '66). Furthermore, the appearance of ondary ossification centers in many mammals novel mesopodial arrangements may have sig- often is correlated poorly with attainment of nificant evolutionary consequences. For exam- maximum adult size. Specifically, in rodents 76 J. HANKEN (Dawson, '29, '34a, b, '35; Strong, '26), guinea of California at Berkeley, during which time pigs (Zuck, '38) and opossums (Washburn, '46) many people provided aid and encouragement. fusion may be delayed until late in life, years Doug Eakins, Tom Hetherington, and H. after the cessation of longitudinal growth. Fur- Bradley Shaffer accompanied me on field trips thermore, a given specimen may demonstrate to Mexico and (except in rare instances) enthu- great regional variation in the timing of epiph- siastically collected Thorius. John Cadle, Car- yseal fusion within the body; in some bones, los Ceron, Julie Feder, Martin Feder, Jim epiphyseal fusion may never occur (Washburn, Lynch, Ted Papenfuss, Bob Seib, David Wake, '46). Second, most birds have only a single Marvalee Wake, and Tom Wake provided addi- epiphyseal ossification center that lies at the tional material. Richard Wassersug helped me head of the tibiotarsus (Bellairs and Jenkin, adapt the double-stain procedure for Thorius; '60; Hinchliffe and Johnson, '80); regulation of Marvalee Wake allowed access to her histology growth of the remaining elements is achieved lab. James Patton, Richard Wassersug, Brian without secondary centers. Thus, although Hall, Marvalee Wake, Sally Susnowitz, and secondary (epiphyseal)ossification centers are two reviewers provided helpful comments and components of the pattern of determinate skel- suggestions on various drafts of this paper. etal growth that is seen in many amniotes, Mary Primrose, Terry Collins, and Phyllis they are not a requisite feature of the mechan- Thompson helped prepare the figures. David ism conferring precise growth and size regula- Wake made the original suggestion that I tion in the entire skeleton in these groups. study Thorius, and contributed enthusiasm, Amphibians typically lack secondary ossifi- logistic support, and valuable intellectual dis- cations in long bones, although calcified carti- cussion throughout my stay at Berkeley. This lage has been described in the epiphyses of sev- research was partially supported at the Uni- eral frogs (Haines, '42). Among plethodontid versity of California, by a Louise M. Kellogg salamanders, adult Thorius are unique in the Grant-in-Aidand an Annie Alexander Memorial extent of appendicular ossification, including Fellowship from the Museum of Vertebrate Zool- long bones and mesopodial elements, although ogy, a Center for Latin American Studies Grant- they too lack secondary centers. This "exces- in-Aid,the Department of Zoology, and National sive" ossification is part of a mechanism of de- Science Fondation grant DEB-7803008to D.B. terminate skeletal growth that confers 1) size Wake; it was completed while I was a Killam reduction relative to larger, generalized pletho- Postdoctoral Fellow at Dalhousie University. dontid salamanders, and 2) adult size differ- ences among species within the genus. How- ever, empirical estimates of growth in natural LITERATURE CITED populations of other plethodontid salamanders Abdalla, 0. (1979)Ossification and mineralization m the ten- suggest that skeletal growth may be quite de- dons of the chicken (Gallus domesticus). J. Anat. 129351 - terminate in these groups as well; in many 359. Alberch, P. (1980)Ontogenesis and morphological diversifi- taxa, such as Batrachoseps attenuatus (Hen- cation. Am. 2001.20.653-667. drickson, '54), rostrata (Houck, Alberch, P. (1981)Convergence and parallelism in foot mor- '77), and Desmognathus ochrophaeus (Tilley, phology in the neotropical salamander genus Bolito- '77, '80),little or no overall size increase is ob- glossa. I. Function. Evolution 35234-100. Alberch, P.. and J. Alberch (1981) Heterochronic mecha- served following the attainment of sexual ma- nisms of morphological diversification and evolutionary turity. The mechanism of skeletal growth regu- change in the neotropical salamander, Bolitoglossa occz- lation and adult size differentiation in these dentalis (Amphibia: Plethodontidae). J. Morphol. 167: groups (and, presumably, other amphibians) 249-264. Andrew, W., and C.P. Hickman (1974) Histology of the Ver- does not involve the conspicuous modifica- tebrates. St. Louis: Mosby. tions of the appendicular skeleton that are Bellairs, A.DA.. and C.R. Jenkin (1960) The skeleton of characteristic of Thorius. These results sug- birds. In A.J. Marshall (ed.): Biology and Comparative gest the existence of a plurality of mechanisms Physiology of Birds. New York: Academic, pp. 241-300. Carroll, R.L. (1977) The origin of lizards. In S.M. Andrews, of growth regulation in tetrapods, involving a R.S. Miles, and A.D. Walker (eds.): Problems in Verte- variety of skeletal modifications. Knowledge brate Evolution. New York: Academic, pp. 359-396. of the full extent of this diversity awaits analy- Cope, E.D. (1869a)A review of the species of the Plethodon- sis of the mode and mechanism of skeletal tidae and Desmognathidae.Roc. Acad. Nat. Sci. Philadel- phia. 1869:93-118. growth, and growth regulation, in other Cope, E.D. (1869b)Natural history miscellany. . . new sala- groups. mander. Am. Nat. 3222. Dawson, A.B. (1929) A histological study of the persisting ACKNOWLEDGMENTS cartilage plates in retarded or lapsed epiphyseal union in the albino rat. Anat. Rec. 43:109-129. This study was undertaken while I was a Dawson, A.B. (1934a) Further studies on epiphyseal union graduate student in zoology at the University in the skeleton of the rat. Anat. Rec. 60:83-86. SKELETAL GROWTH AND VARIATION IN SALAMANDERS 77

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