Cytologia 48: 177-183, 1983 Karyotypes and Evolutionary Relationships of Trionychoid Turtles John W. Bickham, James J. Bull, and John M. Legler Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX, 77843, USA: Population and Developmental Biology Group, School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex, BNL 9QG, England; Department of Biology, University of Utah, Salt Lake City, Utah, 84112, USA. Received March 2, 1981 The families Carettochelyidae (pitted-shelled turtle) and Trionychidae (soft -shelled turtles) are considered closely related (Baur 1891, Romer 1966, Zug 1966, Gaffney 1975, Mlynarski 1976, Pritchard 1979), but there is little agreement con cerning their relationships to other turtles. Webb (1962) concluded that the families Trionychidae, Carettochelyidae, Chelydridae, and Dermatemydidae are related. Gaffney (1975) used similarities in skeletal morphology as the basis for grouping the families Trionychidae, Carettochelyidae, Dermatemydidae, and Kinosternidae in the superfamily Trionychoidea. Serological tests (Frair 1964) indicated that Trionyx is highly divergent from the other cryptodires studied (Carettochelys not included). Cross reactivities between T. ferox and a chelydrid, a kinosternid, an emydid, a testudinid, a cheloniid, a dermochelyid, and a pelomedusid were all about equally low. It has been shown that turtles are conservative in their rate of karyotypic evolution and that variation is found mostly at the subfamily or family level (Bickham and Baker 1979, Bickham 1981). Therefore, karyotypic similarity among higher taxa of turtles may be reflective of evolutionary relationships. We herein report a study of the karyotypes of Carettochelys insculpta and three species of Trionyx and assess the degree of similarity between the karyotypes of Trionyx and Chelonia mydas (as an example of a non-trionychoid turtle) using banded chromosome morphology. Materials and methods Standard karyotypes (nondifferentially stained) were prepared from leukocyte cultures (Bull and Legler 1980) while banded karyotypes were prepared from cultured fibroblasts (Bickham and Baker 1976, Sites et al. 1979b). G-bands were produced by treating air dried slides in a trypsin solution prior to staining in Giemsa (Seabright 1971). C-bands were obtained using air dried slides treated in 0.2M HCl (20min), 5% barium hydroxide (20-60min), 1 X SSC (overnight) and stained in Giemsa (Sumner 1972). Results Chromosomes are arranged in descending order of size in Figs. 1 and 2. The 178 John W. Bickham, James J. Bull, and John M. Legler Cytologia 48 first row of each karyotype is composed of macrochromosomes and the second and third rows are microchromosomes. The distinction between these two groups is arbitrary in taxa whose chromosomes gradually grade in size from large to small. We consider macrochromosomes to be of sufficiently large size that centromere position can be consistently determined even in spreads of mediocre quality. A description of the karyotype of each species is given below. Fig. 1. Standard karyotypes of trionychoid turtles. A , Carettochelys insculpta (2n=68; subadult), B, Trionyx spiniferus (2n=66; •Š) . Carettochelyidae-Carettochelys insculpta, 2n=68, Fig. 1a. The standard karyotype of a single sub-adult specimen consisted of 8 pairs of biarmed macro chromosomes and 26 pairs of microchromosomes . The two largest pairs are me tacentric, the third and fourth pairs are submetacentric , and pairs 5-8 are metacen tric. There is a distinct size gap between pairs 6 and 7 and a slight diff erence between the smallest macrochromosome (pair 8) and the largest microchrom osome (first pair in second row). 1983 Karyotypes and Evolutionary Relationships of Trionychoid Turtles 179 Trionychidae-Trionyx spiniferus, 2n=66, Figs. 1b and 2. G-bands and standard karyotypes from 3 females were examined. The karyotype of this species has 8 pairs of macrochromosomes and 25 pairs of microchromosomes. The first two pairs of macrochromosomes are metacentric, the third is submetacentric, pairs 4 and 5 are acrocentric, pair 6 is subtelocentric, and pairs 7-8 are metacentric. There is a distinct size gap between pairs 6 and 7, but the largest microchromosome (first pair in row 2) is nearly as large as the smallest macrochromosome. Fig. 2. G-band comparison of the macrochromosomes of Chelonia mydas and Trionyx spiniferus. The macrochromosomes of C. mydas are arranged into group A (metacentric or submetacentric) and group B (telocentric or subtelocentric) (Bickham et al. 1980). In all pairs the chromosomes of C. mydas are placed on the right and the presumed homologous chromosome of T. spiniferus, if identifiable, is placed to the left. Fig. 3. G-band karyotype of Trionyx ferox (2n=66; •‰). Fig. 2 is a banding comparison of the macrochromosomes of Chelonia mydas (Cheloniidae) and T. spiniferus. The first two pairs in group A appear unaltered between the two species as do the last two of group B. The third pair in group A is acrocentric in T. spiniferus and metacentric in C. mydas indicating a pericentric inversion. The last four chromosomes of group A and the second of group B in 180 John W. Bickham, James J. Bull, and John M. Legler Cytologia 48 C. mydas are not identifiable in T. spiniferus. The first pair in group B is submeta centric in T. spiniferus and subtelocentric in C. mydas indicating a pericentric inver sion. The third pair in group B differs by the presence of a heterochromatic short arm in C. mydas that is absent in T. spiniferus. Trionyx ferox, 2n=66, Fig. 3. Standard karyotype and G-bands were studied from a single male. No differences were detected between the karyotype of this individual and that of the three Trionyx spiniferus. Trionyx sinensis, 2n=66. Standard karyotypes and C-band preparations were obtained for one specimen (subadult) and a standard karyotype from a second specimen (male). No differences were detected between the karyotype of this species and those of T. spiniferus and T. ferox. C-bands are restricted to the centromeric regions as is characteristic of many turtles (Bickham and Baker 1976, Bull and Legler 1980). Discussion Chromosomal evolution is highly conservative in cryptodiran turtles, most species within a family or subfamily possess identical karyotypes (Bickham and Baker 1976, 1979, Stock 1972). Extensive homology of chromosomal banding patterns has been demonstrated even between such divergent groups as Emydidae and Cheloniidae (Bickham et al. 1980). The karyotypes of Carettochelys and Trionyx are similar in possessing a higher diploid number than other cryptodiran species (Table 1). Thus, a high degree of homology between Carettochelys and Table 1. Reported diploid numbers of trionychoid turtles. A question mark after the diploid number indicates a possibly inaccurate report Trionyx is inferred from the general similarity of the nondifferentially stained karyo types of the two species. Karyotypes of Carettochelys and Trionyx differ from those of other cryptodires. The macrochromosomes of Chelonia mydas (Bickham et al. 1980) are compared to their presumed homologues in Trionyx spiniferus (Fig. 2). All but 4 of the 12 pairs 1983 Karyotypes and Evolutionary Relationships of Trionychoid Turtles 181 of macrochromosomes have been rearranged since the divergence of Chelonia and Trionyx from a common ancestor. This contrasts markedly with the high degree of homology found among all other families of crytodires for which banding data are available (Bickham 1981, Bickham and Baker 1976, 1979, Bickham et al. 1980, Haiduk and Bickham 1982, Sites et al. 1979a, b). It cannot be proven which taxon (Chelonia or Trionyx) has remained primitive and which has become derived, or whether they both diverged from an ancestor with some intermediate karyotype. However, this may be resolved when the primitive karyotype of the Pleurodira is known. Bull and Legler (1980) hypothesized the primitive diploid number for the Pleurodira to be 2n=50-54 because of the similarity of standard karyotypes in many cryptodires and pleurodires that have diploid numbers in this range. This would suggest that the karyotype of Chelonia could be ancestral to that of the trionychoids. Table 1 lists reported diploid numbers for trionychid and carettochelyid turtles. Approximately one-third of the recoginzed trionychid species have been karyotyped, including members of both subfamilies (Lissemyinae and Trionychinae). The reported diploid numbers other than 2n=66 are questionable because in all cases more recent studies using modern cytological techniques have failed to substantiate them. The 2n=52-54 report for Trionyx leithii (Singh et al. 1970) was actually the karyotype of an emydid turtle (Singh 1972) so no karyotypic data are available for this species. From the above, we infer that the primitive karyotype of triony chids was 2n=66 and that the common ancestor of trionychids and carettochelyids had a diploid number close to this. Thus, two points are evident: 1) standard karyotypes of Carettochelys and Trionyx are similar, and 2) the karyotypes of these two genera are considerably different from the karyotypes of all other cryptodiran turtles. If the high diploid number of trionychoids is derived, then it is likely that Carettochelys is closely related to the Trionychidae. It is improbable that these similarities would be independently derived, considering that karyotypic differences are otherwise slight among cry ptodires. Should the trionychoid karyotype be ancestral and the karyotypes