JOURNAL OF MORPHOLOGY 190:191-200 (1986) Native Variant Limb Skeletal Patterns in the Red-Backed Salamander, Plethodon cinereus, are not Regenerated CHARLES E. DINSMORE AND JAMES HANKEN Department of Anatomy, Rush Medical College, Chicago, Illinois 60612, The Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672 (C.E.D.), and Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, Colorado 80309- 0334 (J. H.) ABSTRACT Species of the salamander genus Plethodon have a character- istically uniform morphology. Morphological conservatism at the level of inter- specific comparisons, however, is not always reflected within species. Perhaps the most extreme example of intraspecific variation is the recent description of extensive variability in limb-skeletal patterning both within and between populations of the widespread species P. cinereus. We utilized limb regenera- tion following experimental amputation as a tool 1) to examine whether natu- rally occurring variant skeletal patterns result from limb loss and regeneration in nature, and 2) to assay the intrinsic (i.e., genetic) component of between- individual variation in mesopodial patterning. We observed the following. First, regenerate patterns are strikingly different from native patterns: inter- element fusions in regenerates are typically between proximodistally adjacent cartilages, whereas interelement fusions in native variant limbs occur exclu- sively between laterally adjacent cartilages. Fusions also are over ten times more frequent in regenerates than in native limbs. Second, there is no strong correlation between native limb pattern (typical vs. variant) and the regener- ate pattern. We conclude that variability in field-collected P. cinereus reflects extensive intrapopulation variation in limb-skeletal patterning during origi- nal limb development, rather than regeneration in nature, and that limb regeneration analysis provides no evidence of a strong genetic component to between-individual variation. Finally, unusual mesopodial patterns produced during limb regeneration may be related to the mechanical factors impinging on the regenerating limb in this terrestrial species. Urodele species of the genus Plethodon have a remarkably uniform morphology, es- pecially in terms of osteology (Wake, '63). Indeed, the genus has been offered as a par- adigm of morphological stasis among verte- brates (Wake et al., '83). Recently, however, a Nova Scotian population of the wide-rang- ing species P. cinereus was found to contain an exceptionally high incidence and diver- sity of variant mesopodial (i.e., wrist and an- kle) and digital skeletal patterning. The variant mesopodial patterns comprise differ- ent fusion combinations between adjacent cartilages; the variant digital patterns typi- cally involve a reduced number of phalanges in one or more digits. Some of these patterns are found in other plethodontid genera (Han- ken, '83, '85; Wake, '661, but other, less fre- quent patterns are unique to that population. The significance of this variability is under- scored by noting that certain variant pat- terns in this population are as different as those that distinguish among other urodele species, or even genera. Intraspecific skeletal variation in Plethodon is not always as "con- servative" as that between species (Hanken, '83). We subsequently quantified limb skeletal variation in three other, geographically dis- tant populations of P. cinereus from both within and outside Nova Scotia and found that variation is indeed widespread (Hanken and Dinsmore, '86). While the frequency of mesopodial variants in Nova Scotia is two to three times higher than that found else- where-and involves a greater diversity of Address reprint requests to Charles E. Dinsmore, Department of Anatomy, Rush Medical College, 600 South Paulina Street, Chicago, IL. 60612. 0 1986 ALAN R. LISS, INC.192 C.E. DINSMORE AND 3. HANKEN skeletal patterns-variability is also present in populations as far away as Maine and Virginia; in these latter populations, the fre- quency of variant patterns in the carpus alone exceeds 10%. It is also important to note that, while their exact frequencies vary, the predominant patterns in each population are the same. There are several possible explanations for this osteological variation; each may fall un- der a broadly defined principal category of either hereditary or environmental causes. In this study, we explore directly one possible environmental cause: that variant skeletal patterns in the limbs of field-collected ani- mals (here termed native limbs) are in fact regenerated patterns that formed following loss of the original limb in nature. If this hypothesis is correct, then experimentally amputated limbs with the native typical (i.e., most common) skeletal pattern should pro- duce regenerates with the naturally occur- ring typical or variant patterns. In addition, and based upon the previous findings that repeated limb amputation in newts increases the incidence of abnormal regenerates (Dear- love and Dresden, '76), limbs that originally bore variant skeletal patterns should pro- duce regenerates with a higher incidence and possibly wider range of variability than oc- cur among regenerates from native typical limbs. In testing the hypothesis that the native variant skeletal patterns are produced dur- ing regeneration, we are also examining qualitatively the genetic component of be- tween-individual pattern variation. For ex- ample, if the genetic component of variation is relatively high, then patterns regenerated from variant limbs should differ from those regenerated from typical limbs. Alterna- tively, if the genetic component is relatively low, then the frequencies and types of pat- terns regenerated should not differ signifi- cantly between native typical and native variant limbs. Another possible result of experimental limb amputation is that regenerated pat- terns are qualitatively different from both native typical and native variant patterns. A substantial body of evidence, however, sup- ports the claim that both original develop- ment and regeneration of urodele limbs are guided by the same basic mechanisms (e.g., reviews by Faber, '71; Muneoka and Bryant, '82, '84; Stocum, '75; Stocum and Fallon, '82). Furthermore, the mesopodia, on