C 1996 The Japan Mendel Society Cytologia 61: 169-178, 1996 Cytotaxonomic Characterization of the Genera Scleratoscopia and Tetanorhynchus (Orthoptera-Proscopiidae) R. C. Mour1 M. J. Souza1 and T. Tashiro2 1 Departamento de Genetica (CCB), 2Departamento de Educacao Fisica (CCS), Universidade Federal de Pernambuco, Recife-PE, 50732-970 Brazil Accepted March 13, 1996 Proscopiidae is an endemic family in South America and is considered to descend from acridomorphs or pre-acridomorphs inhabiting the South American region of Gondwana (Carbonell 1977). From a taxonomic viewpoint, proscopiids are quite distinct from other acridomorph families. However, some similarities in external morphology and phallic complex are detected when this family is compared to Eumastacidae, especially the Australian subfamily Morabinae. This evidence suggests that these families are phylogenetically related (Dirsh 1961, Randell 1963, Blackith and Blackith 1966). According to Amedegnato (1993), the similarities between morabines and proscopiids indicate that these groups are members of the same phyletic unit although descending from different eumastacids. Analysis of the structure of male genitals and of other traits indicates that proscopiids are derived from an ancestral group of Eumasta- cidae (Cryptophalli), the Teicophrynae subfamily of North America. The members of this subfamily present a structure of the phallic complex similar to that of Asian Gomphomastaci- nae, which probably gave origin to morabids. On the basis of a study of male genitals, Jago (1989) extensively reviewed the taxonomy of the family Proscopiidae. In this analysis, he reviewed and defined 21 genera, seven of them new (Astromascopia , Bolichohynchus , Carphoproscopia , Microcoema , Pseudastroma , Scopaeoscle- ratoscopia and Scleratoscopia). The description of the genus Scleratoscopia (Jago 1989) was based on the species type Cephalocoema protopeirae Amedegnato 1985 and comprises three species: S. protopeirae, S. spinosa and S. silvai. Rehn (1957) had previously described the last species as Tetanorhyncus silvai. Few reports are available on the chromosomes of Proscopiidae. Piza (1943, 1945) described the first karyotypes of this family in a study of the species Cephalocoema zilkari and Tetanorhynchus mendesi, with 2n = 17, XO ( •‰ ). This family is characterized by diploid numbers of 2n =19, 2n =17 and 2n = 15, with a predominance of acrocentric chromosomes and mechanisms of sex determination of the XO : XX type (Piza 1943, 1945, Dasgupta 1968, Mesa 1973, Cea 1974, Ferreira 1978, Mesa and Ferreira 1981). No chromosomal study has been carried out thus far on the family Proscopiidae using differential staining techniques, in contrast to what occurred with Acrididae (King and John 1980, Santos et al. 1983, Rufas et al. 1985, Suja et al. 1991), a family in which countless species have been analyzed both by C-banding and by silver staining. These techniques have permitted a better characterization of karyotypes and analysis of the phylogenetic relations between species. In the present study, the species Scleratoscopia protopeirae Amedegnato 1985, S. spinosa Jago and S. silvai (Rehn) comb. n. made by Jago (1989) were analyzed comparatively in terms of standard karyotype, C-banding pattern and nucleolar chromosomes. These species were also investigated in terms of external morphology and structure of the phallic complex. This last analysis suggested the reassignment of silvai to the genus Tetanorhynchus, as initially described by Rehn (1957). Thus, in the present study we considered the description of Rehn (1957). 170 R. C. Moura, M. J. Souza and T. Tashiro Cytologia 61 Material and methods The specimens studied in the present investigation were collected from the rural region and from the "caatinga" of the State of Pernambuco, Northeast Brazil. Males and females of three species collected at different sites were analyzed cytogenetically: S. protopeirae (56 individuals: Gravata), S. spinosa (23 individuals, 4 from Pesqueira, 12 from Ouricuri and 7 from Arari- pina), and Tetanorhynchus silvai (57 individuals; 3 from Joao Alfredo, 38 from Arcoverde and 16 from Serra Talhada). Cytologic preparations were obtained by the classical squashing technique and the chromo- somes were stained with 1% lactoacetic orcein. All females were treated with colchicine (0.1 % in insect saline) at the proportion of 0.1 m1/3 g body weight. Testis and ovaries were fixed in 3 : 1 ethanol : acetic acid. C-banding was performed by the technique of Sumner (1972) with modifications (the slides were treated with 0.2 N hydrochloric acid for 30 min at room temperature, followed by a 5% barium hydroxide solution at room temperature for 20 to 30 min and by 2 X SSC at 60°C for 45 min). Silver nitrate staining was performed by the method A A' s C Fig. 1. Phallic complex of the Tetanorhynchus silvai, Scleratoscopia protopeirae and S. spinosa. A, dorsal aspect and A', lateral aspect from phallic complex; B, lateral plates; C, Epiphallus. (1) transverse plate; (2) "lophi"; (3) lateral plate. 1996 Cytotaxonomic Characterization of Scleratoscopia and Tetanorhynchus 171 of Rufas et al. (1987). Slides were treated with 2 X SSC heated to 60°C for 10 min and then stained with silver nitrate (0.1 g Na3Ag per 0.1 ml distilled water plus formic acid), pH 3.5. Genitals were analyzed on the basis of drawings made under a magnifying glass with 6.4 X magnification on which a millimeter grid had been placed. We measured 12 traits in 25 male specimens of each species with the aid of a pachymeter (Table 1). Data were analyzed statistically (One-way analysis of variance) and variables such as the mean, standard deviation and variance were calculated. The Tukey test was used, with the level of significance set at 1%. Copex Pan film Agfa (ASA 12.5) was used for the photomicrographs, and Brovira Agfa 3 paper was used for the photographic copies. The specimens studied in the present investigation are deposited in the insect collection of the Department of Genetics, UFPE, Recife, PE, Brazil. Results Structure of the phallic complex and external morphological traits After dissection, the structures of the genitals from the three species were analyzed comparatively for a better observation and detection of differences between species. These structures were strongly sclerotized in S. protopeirae and S. spinosa, with differences concentrat- ed in the lateral plates. These plates have small spines throughout the lobes and along the crevices which are detected in larger quantities in S. spinosa. The apices of the lateral plates were flat in S. protopeirae and projected upward in S. spinosa. The genitals of S. silvai (=T. silvai), presented on the other hand a basic pattern corresponding to the genus Tetanorhynchus, with intermediate sclerotization and without projection of the lateral plates (Fig. 1). The structural differences observed between genitals confirm the identity of the three species at the species level and suggest that S. silvai belongs to the genus Tetanorhynchus, as proposed by Rehn (1957). Comparative analysis of twelve external morphological traits was carried out on 25 male specimens per species. Head length and width, fastigium, interocular distance, femur, pro- Table 1. Comparative data concerning twelve morphological traits of Scleratoscopia protopeirae (Sp), S. spinosa (Ss) and Tetanorhynchus silvai (Ts) The mean (.-x"),standard deviation (s), variance (F) and the results of analysis of the difference between means (D.t) using the Tukey test at 1% level of significance are presented for the twelve traits studied: HEA = head; FAS = fastigium; IOC = interocular distance; FEM = femur; PRO = pronotum; MES = mesothorax; MET = meta- thorax; TIB = tibia. The letters I and w following the abbreviations represent length and width, respectively; = Significant difference. 172 R. C. Moura, M. J. Souza and T. Tashiro Cytologia 61 notum, mesothorax, metathorax, and tibia were measured. One-way analysis of variance showed that the three species differ from one another in at least one of the means for the twelve traits. The Tukey test at the 1% level of significance showed that five traits (fastigium, interocular distance, mesothorax, metathorax and tibia) presented significant differences among the three species (Table 1). Chromosome complement The three species analyzed showed a diploid number of 2n = 19 (•‰) and 20 ( •¬ ) and a sex-determining mechanism of the XO type. Fig. 2 presents a karyogram for each species obtained from cells of the ovariole wall. The karyotypes of S. protopeirae and S. spinosa consist of three pairs of large chromosomes (L1—L3), three pairs of medium chromosomes (M4—M6) A B C Fig. 2. Somatic chromosomes of females of Proscopiidae. Tetanorhynchus silvai (A); Sclerato- scopia protopeirae (B) and S. spinosa (C). 1996 Cytotaxonomic Characterization of Scleratoscopia and Tetanorhynchus 173 and four pairs of small chromosomes (S7—S10). In T. silvai, all chromosomes were acrocentric, with a gradual increase in size. Chromosomes 1, 3 and X were submetacentric in the two Scleratoscopia species and the remainig ones were acrocentric. In all three species, chromosome X presented positive heteropycnosis at the beginning of prophase I of meiosis and negative heteropycnosis in metaphase I (Fig. 3). The C-banding pattern observed in the three species revealed the presence of small pericentromeric bands in all chromosomes in the complement. Pairs 7 and 8 and chromosome X of T. silvai had C bands of larger size (Figs. 4, 5).