C 1996 The Japan Mendel Society Cytologia 61: 169-178, 1996

Cytotaxonomic Characterization of the Genera Scleratoscopia and Tetanorhynchus (-)

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 , 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 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 (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 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). In all three species, silver nitrate staining of primary spermatocytes, although showing nucleolar remnants of irregular shape and deeply stained in prophase I, was inconclusive with respect to the precise identification of nucleolar organizer chromosomes. Staining of spermat- ids from S. protopeirae and T. silvai permitted the identification of two nucleolar masses in 50% of the cells analyzed, suggesting that these species have at least two nucleoli, one of which is probably associated with chromosome X (Fig. 6). The kinetochore regions stained differently in the three species and were more deeply labeled in T. silvai.

Discussion

The three species analyzed in the present study are quite similar, although they can be

B

A C

Fig. 3. First metaphase of Scleratoscopia protopeirae (A), Tetanorhynchus silvai (B) and S. spinosa (C). X chromosome is negatively heteropycnotic. 174 R. C. Moura, M. J. Souza and T. Tashiro Cytologia 61

Fig . 4 . C-banded metaphase I of Scleratoscopia spinosa (A), partial diplotene of S. protopeirae (B) and spermatogonial metaphase of Tetanorhynchus silvai (C). In C the large heterochromatic blocks of the bivalent 7 and 8 are indicated by arrows.

A

B

C Fig . 5 . Idiograms showing the C-banding patterns. Tetanorhynchus silvai (A), Scleratoscopia protopeirae (B) and S. spinosa (C). 1996 Cytotaxonomic Characterization of Scleratoscopia and Tetanorhynchus 175

A B

Fig. 6. Silver-stained spermatocytes from Tetanorhynchus and Scleratoscopia. Spermatides of T silvai (A); Early pachytene of S. spinosa (B). Nucleolus indicated by arrows. distinguished by analysis of the phallic complex. The study of the male genitals of this species permitted the observation of significant difference with respect to the lateral plates; the absence of a projection of the apex is S. silvai suggests that this species may not belong to this genus. Considering the taxonomic descriptions of Rehn (1957) and Jago (1989) and the fact that the latter author did not examine the species type T. punctatus Klug, 1820 in his analysis of the genus Tetanorhynchus, it is possible that S. silvai belongs to the genus Tetanorhynchus. Comparative study of the external morphological traits of the species S. protopeirae, S. spinosa and T. silvai showed that traits such as fastigium, interocular distance, mesothorax, metathorax and tibia are also important differential taxonomic parameters for the analysis of these species. These five traits differed statistically among species. The species studied in the present investigation are being described for the first time in chromosomal terms. The diploid number of 2n =19 detected in Scleratoscopia and Tetanor- hynchus is predominantly observed in other genera of the family Proscopiidae. This is the case for Cephalocoema which presents nine species with this karyotype, and for four other species of Tetanorhynchus (Mesa and Ferreira 1981). Of the 29 species of proscopiids analyzed thus far, 55.17% have karyotypes with 2n = 19, 37.93% have karyotypes with 2n = 17 and 6.89% have karyotypes with 2n = 15. Mesa and Ferreira (1981) proposed a model to explain the karyotypic evolution of this family. In this model, Morabinae (Australian) and Proscopiidae (South American) are assumed to derive from a common ancestral chromosome complement with 2n =17 consisting of six pairs of acrocentric chromosomes and two pairs of metacentrics, with a fundamental number equal to 21. The Proscopiidae species Hybusa armaticolis represents this karyotype, which is considered to be primitive. Different sequences of chromosome rearrangements with fusion, dissociation and pericentric inversion have been proposed to explain the diversity of karyotypes (2n =19 to 2n =15) detected in Proscopiidae. The occurrence of acrocentric chromosomes predominates in the karyotypes with 2n =19, 2n = 17 and 2n = 15. Only in three species of Cephalocoema and one of Tetanorhynchus were 2n = 17 karyotypes detected with one pair of submetacentric autosomes, whereas Hybusa armaticolis (2n =17) and two species of Anchocoema (2n =15) presented two submetacentric 176 R. C. Moura, M. J. Souza and T. Tashiro Cytologia 61 pairs (Mesa and Ferreira 1981). Of the three species analyzed here, only T. silvai presented the common karyotype of the family consisting solely of acrocentrics. S. protopeirae and S. spinosa have two pairs of submetacentric autosomes. These two species with karyotypes of 2n = 19 and biarmed chromosomes represent the first cases of pericentric inversions knows thus far in this type of karyotype. Heterochromatin segments, which are responsible for large part of the polymorphisms detected in natural populations of Acrididae, have not been detected frequently in morabines and prosecopiids. The first report about the pattern of constitutive heterochromatin distribu- tion, presented here, shows that the three species analyzed have a small amount of constitutive heterochromatin represented by small pericentromeric blocks. The polymorphic condition is the situation most frequently detected for pericentric inversions in natural populations. Genera such as Trimerotropis (Oedipodinae) and Moraba (Morabinae) are examples of the fundamental role played by this type of rearrangement in karyotypic evolution (White 1951, 1957). Scleratoscopia presented mono- morphism for the inversions that involve two autosome pairs and chromosome X in S. protopeirae and S. spinosa. The absence of polymorphism in this genus may represent a primitive condition or indicate that these inversion represent derived and fixed karyotypes, or yet it may be due to the small number of specimens analyzed thus far in each population. No case of inter or intraspecific polymorphism has been described thus far for the family Proscopiidae. Data about the family Acrididae, especially Gomphocerinae (Rufas et al. 1985, Cabrero and Camacho 1986) and about the family (Rocha 1995) show that silver nitrate stains the nucleoli, NOR, synaptonemal complex, cores, kinetochores and adjunct centrioles in a different manner. Our data show that, in contrast to what occurs in the families Acrididae and Romaleidae, the use of silver staining in spermatocytes of Proscopiidae did not permit the observation of other nuclear structures, but only labeled nucleoli and kinetochores. This is probably due to the structural chromosome differences existing between proscopiids and the other two families of acridomorphs. However, these data represent only a preliminary analysis of NOR distribution in these organisms. Cytogenetic studies and analysis of the external morphology and of the phallic complex of the species Tetanorhynchus silvai Rehn, 1957 (=Scleratoscopia silvai (Rehn) comb. n. Jago, 1989), Scleratoscopia protopeirae Amedegnato, 1985 and S. spinosa Jago, 1989 showed signifi- cant differences between these taxa, reinforcing their identities at the species level. These data suggest that S. silvai Jago, 1989 should be reassigned to the genus Tetanorhynchus according to the original description made by Rehn, 1957.

Summary In the present investigation we studied cytotaxonomically three species of of the family Proscopiidae, Tetanorhynchus silvai, Scleratoscopia protopeirae and S. Spinosa. Analysis of the phallic complex and of external morphological traits showed significant differences between species. In particular, comparative analysis of the phallic complex suggested that Scleratoscopia silvai Jago, 1989 should be reassigned to the genus Tetanorhyn- chus, as first described by Rehn (1957). T. silvai, S. protopeirae and S. spinosa have diploid numbers of 2n =19 X0 in males and 2n = 20 XX in females. The chromosomes of these species are described here for the first time. Although they present the same diploid number, these species differ in chromosome morphology. T. silvai showed a karyotype consisting solely of acrocentric chromosomes, whereas S. protopeirae and S. spinosa have karyotypes formed by two 1996 Cytotaxonomic Characterization of Scleratoscopia and Tetanorhynchus 177

pairs of submetacentric autosomes and seven pairs of acrocentrics. In these two species, chromosome X is submetacentric. The karyotype of Scleratoscopia was considered to be derived due to the probable occurrence of pericentric inversions from an ancestral karyotypes

consisting of acrocentrics. Three species analyzed are monomorphic for their karyotypic constituints. C-banding showed that these species have small blocks of constitutive heterochro-

matin in all chromosomes in the complement, except for pairs 7 and 8 and for chromosome X of T. silvai, which presented larger blocks. Silver nitrate staining in early prophase and in

specrmatids showed the occurrence of at least two nucleoli for each species studied. S. protopeirae and S. spinosa represent the first species in the family Proscopiidae, with a karyotype of 2n =19 (•‰) in which biarmed chromosomes occur. The chromosomal differences between T. silvai and the Scleratoscopia species also support the reassignment of S. silvai to the genus

Tetanorhynchus.

Acknowledgments

We are grateful to Dr. Carlos S. Carbonell for the taxonomic identification of the species studied and to Dr. Marcelo Guerra for revising the manuscript and for helpful suggestions. This research was financed by Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE) and by Conselho Nacional de Desenvolvimento Cientifico a Tecnolo- gico (CNPq).

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