Cytological procedure
Material examined
Cytological material used in this study was prepared over several years, ranging from 1964 to 1971, and under a variety of laboratory conditions, therefore preparation techniques have varied considerably during the course of the study. However, most chromosome spreads were made using basic techniques similar to those of Evans et al. (1964), Patton (1967a), Bianchi and Contreras (1967), Hsu and Patton (1969), and Ford and Evans (1969). A few preparations were made with the temporary aceto-orcein squash technique (Darlington and Ie Cour, 1962) and in rare instances these were preserved with the dry-ice-quick-freeze procedure. Tissues examined were generally testes, from sexually active males, and bone marrow and spleen from females. In non-reproductive males all three tissues were generally used. To minimize preparation time and maximize the number of individuals examined, all tissues from a given animal were generally mixed together in a single cell suspension. To further maximize productivity, two to four preparations were processed in parallel and earlier and later stages of three parallel sets were overlapped. By using these expedients, the average time used in processing an individual from sacrifice to preliminary karyotype determination was usually kept to less than a half hour.
To allow rapid feedback to the collecting program and to insure that the animals were in close to optimal condition when karyotyped, most preparations were made in the field, generally in convenient motels or in laboratory space kindly provided by local institutions (see acknowledgments). Unfortunately, since some preparations were made under primitive conditions and the microscope used to control their quality was not especially good, not all are adequate to resolve the details of microchromosomal morphology. Also, Sceloporus grammicus from the Teotihuacan area of the Valley of Mexico were generally so wary that many had to be killed for capture. These were immediately placed on crushed ice in a styrofoam container and were usually processed within six hours. In all cases the killed lizards could be unequivocally scored as to which karyotype population they belonged, However, because they did not benefit from a colchicine pretreatment, and because of the inevitable (but surprisingly slow) cell degradation, the microchromosomes could not always be counted, nor was it always possible to tell which of the macrochromosomes in fissioned karyotypes retained the metacentric condition. Where these technical problems are pertinent they will be mentioned. Most preparations were preliminarily scored as aceto-orcein stained wetmounts in the field. Counts of chromosome number were generally made from at least three good spreads from each animal. In reproductive males, counts were usually made from diakinesis figures, but frequently at least one mitotic figure was found to check for obvious shifts in centromere positions. Slides were then washed clean with methanol and stored dry and unmounted until they were permanently mounted in the home laboratory. For cases where preliminary determinations were not made, were questionable, or were particularly critical (as in the contact zone study by Hall and Selander, in press), the slides were re-examined in the home lab and usually at least three more good figures checked and their coordinants on the slide recorded. In many cases slides from other animals were also re-examined, and frequently more than the minimum number of figures were inspected.
For detailed comparisons of karyotypes and for preparation of the figures presented here, photographs were taken with either the planapo-brightfield or -phase contrast 100X objectives of a Zeiss Ultraphot II, generally using 4" x 5" Kodak 4154 Contrast Process Ortho film developed in D-11. All photographs used for karyotypes were printed at 3000X (as determined by calibration with a stage micrometer) on Kodabromide paper chosen and processed to provide maximal use of the grey scale. Where feasible, karyotypes were photographed for several individuals of each species and usually represent all of the available subspecies in my material. For some individuals, several karyotypes were prepared to allow intra-individual variation to be compared with inter-individual variation. This is especially important for comparisons of microchromosomal morphology, where much of the observed variation is clearly preparation artifact.
Detailed locality data for all karyotyped specimens except Sceloporus grammicus are presented in the Appendix. Detailed localities for grammicus will be found in Hall and Alvarez (in prep.) and are mapped there and in Hall and Selander (in press) . Here I simply list the number of grammicus belonging to each karyotype population examined from each state. Species are arranged in the Appendix by species groups following Smith and Taylor (1950) as emended by more recent publications and the present work. All species and subspecies are listed, whether karyotypic data exist or not. The following format is used for each locality entry: state name in caps, airline distance from a principal town listed in an excellent topographic road atlas (Caminos de Mexico, 3rd ed., Compania Hulera Euzkadi--B. F. Goodrich, Mexico, D.F. 1967), the elevation in meters, and the Universal Transverse Mercator grid reference coordinants taken directly from the 1:500,000 sheets of the Carta Geografica de la Republica Mexicana, Primera Ed. (pub. by the Comision Intersecretarial Coordinadora del Levantamiento de la Carta Geografica de la Republica Mexicana, Tacubaya, D.F.). This is then followed by the number of specimens examined from that locality. In general, particularly where they are near roads or other surveyed features, the grid references are probably accurate to ±1 kilometer. Compass distances given are only approximate.
In the Valley of Mexico, most localities were plotted on the 1:25,000 topographic maps of the Departamento Cartagraphico Militar, in some cases checked by reference to stereoscopic aerial photographs, both kindly made available through the Departamento de Prehistoria of the Institute Nacional de Antropologia e Historia. For work in the Archeological Zone at San Juan Teotihuacan, Dr. Rene Millon kindly allowed the use of his exceptionally detailed 1:6000 topographic maps (Millon, 1970).
Species were identified following Smith and Taylor (1950), Smith (1936, 1939), or by more recent taxonomic works where appropriate. In some cases identifications have been confirmed by Smith himself. Specimens not preserved frozen for future biochemical studies or used by Hall and Selander (in press) are entered in the herpetological collections of the Museum of Comparative Zoology or Southern Illinois University, Edwardsville.