2013.

The Journal of Arachnology 41:222–226

SHORT COMMUNICATION

Meiotic studies in Brachistosternus alienus (Scorpiones; Bothriuridae)

Renzo S. Adilardi1, Andrés A. Ojanguren-Affilastro2,3,4, Sergio G. Rodrı́guez Gil1, Cristina L. Scioscia2, and
Liliana M. Mola1,3: 1Laboratorio de Citogenética y Evolución, Departamento de Ecologı́a, Genética y Evolución,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, (C1428EHA) Ciudad
Autónoma de Buenos Aires, Argentina; 2División Aracnologı́a, Museo Argentino de Ciencias Naturales ‘‘Bernardino
Rivadavia’’ (CONICET), Avenida Ángel Gallardo 470 (C1405DJR) Ciudad Autónoma de Buenos Aires, Argentina;
3
Carrera del Investigador (CONICET)

Abstract. Brachistosternus Pocock 1893 is the most diverse genus of the scorpion family Bothriuridae. Only four species of
the genus have been cytogenetically analyzed so far. We report herein the cytogenetic analysis of Brachistosternus alienus
Lönnberg 1898 from Comallo (Rı́o Negro province, Argentina). This species is widely distributed in the Monte
phytogeographic province, located in central and northern Argentine Patagonia. Meiotic cells of B. alienus from Comallo
show 23 homomorphic achiasmatic bivalents. The karyotype of this species contains scarce AT-rich regions that may be
associated with the heterochromatin of centromeric regions. Giacomozzi (1977) reports n 5 14 for B. alienus from Chubut
province. Unfortunately, it is not presently possible to determine if those specimens correspond to B. alienus or to a
sympatric species, Brachistosternus angustimanus Ojanguren-Affilastro & Roig-Alsina 2001. These different chromosome
numbers of the two populations analyzed may reflect the occurrence of a chromosomal polytypism in B. alienus, or they
may characterize different species.
Keywords: Cytogenetics, DAPI staining, karyotype, meiosis, scorpion

In recent years, the aim of our research group has been to study are no data on the cytogenetics of this species except for the
cytogenetics in scorpions belonging to the family Bothriuridae Simon chromosome number of specimens from the Patagonian province of
1880. The genus Brachistosternus Pocock 1893 is of particular concern Chubut (Giacomozzi 1977); however, this information is incomplete
because its species-level taxonomy is fairly well resolved (Rodrı́guez Gil and species identity of these specimens is uncertain.
et al. 2009). Moreover, the phylogeny of this group has been recently In this study, two males of B. alienus belonging to a population
evaluated (Ojanguren-Affilastro & Ramı́rez 2009), providing a more from a locality near Comallo (Rı́o Negro province) in northern
comprehensive evolutionary-based framework for cytogenetic studies. Argentine Patagonia (41u039010S, 70u26951.70W) were examined.
Brachistosternus is the most diverse genus of the family Bothriur- Comallo is located about 500 km from the type locality in Chubut
idae, comprising 40 of the ,150 known species of the family. Its province. The karyotype and meiotic behavior of chromosomes was
species inhabit arid and semi-arid areas of western and southern described and furthermore, the distribution of the AT-rich regions in
South America, from Ecuador to southern Patagonia, Argentina a representative of the genus Brachistosternus was determined for the
(Cekalovic 1969; Ojanguren-Affilastro 2001, 2003a, b, 2005a, b; first time.
Ochoa & Ojanguren-Affilastro 2007; Ojanguren-Affilastro & Scioscia The males of B. alienus were collected at night using UV lamps,
2007; Ojanguren-Affilastro et al. 2007a, b). The group’s tremendous then were carried to the laboratory and killed by cooling to 220uC.
diversification is relatively recent, and may have resulted from the Their gonads were dissected in saline solution (0.154 M NaCl),
onset of aridity in the late Miocene (7–10 mya); most of the basal incubated in hypotonic solution (1:1 saline solution:distilled water,
bothriurid genera occur in mesic environments (Prendini 2003). 30 min), fixed in a freshly prepared mixture of ethanol:chloroform:a-
To date, there are few published cytogenetic studies on bothriurids, cetic acid (6:3:1, 30 min), and stored in fresh fixative mixture. Pieces
comprising only ten species (Piza de Toledo 1947; Ferreira 1968; of testis were placed on slides and dissociated in a drop of 80% acetic
Giacomozzi 1977; Rodrı́guez Gil et al. 2009; Schneider et al. 2009a). acid with tungsten needles. Preparations with a drop of suspension
Four of these species belong to the Argentine representatives of the were placed on a heated histological plate (approximately 45uC); the
genus Brachistosternus. Brachistosternus (Ministernus) ferrugineus suspension was spread on the slides using a tungsten needle. Finally,
(Thorell 1876) and Brachistosternus (Brachistosternus) montanus Roig the preparations were air-dried and stained with 5% Giemsa solution
Alsina 1977 have n 5 23 (Rodrı́guez Gil et al. 2009). Brachistosternus in distilled water for 10 min. Following light microscopy observa-
(B.) pentheri Mello-Leitão 1931 shows two different cytotypes, n 5 23 tions, the slides were destained using a mixture of ethanol:acetic acid
and n 5 21, which correspond to two slightly different morphs (3:1) for 2 h and stained with fluorescent dye DAPI (49-6-diamidino-
(Rodrı́guez Gil et al. 2009). Finally, the haploid karyotype of 2-phenylindole) to detect blocks of AT-rich regions. Briefly, the slides
Brachistosternus (B.) alienus Lönnberg 1898 is reported to consist of were rinsed twice with 4xSSC buffer for 10 min and air-dried. One
14 chromosomes (Giacomozzi 1977). drop of 0.5 mg/ml DAPI in PBS (phosphate buffered saline)
Brachistosternus alienus is widely distributed in the Monte containing 1% Triton X-100 was placed on each slide, covered with
phytogeographic province, located in central and northern Argentine a coverslip and incubated in a moist chamber (30 min, room
Patagonia. A taxonomic revision of this species (Ojanguren-Affilastro temperature). Following coverslip removal, the slides were rinsed with
2001) revealed that geographically distant populations exhibit some 4xSSC and air-dried. Finally, they were mounted with 50 ml of
morphological differences, raising doubts about their identity. There VECTASHIELDH (Vector Laboratories, Inc.), covered by a cover-
slip, and stored at 4uC overnight before microscopic analysis. The
4
Corresponding author. E-mail: andres.ojanguren@gmail.com staining with DAPI and air-drying steps were performed in the dark.

222
ADILARDI ET AL.—MEIOSIS OF BRACHISTOSTERNUS ALIENUS 223

Figure 1.—Spermatogenesis, meiotic karyogram and ideogram of Brachistosternus alienus (2n 5 46, n 5 23). a. Early spermatogonial
metaphase; b. Zygotene; c. Pachytene; d. Early postpachytene; e. Middle postpachytene; f. Late postpachytene; g,h. Prometaphase I; i.
Metaphase I; j. Meiotic karyogram (based on prometaphase bivalents depicted in 1g); k. Meiotic idiogram. The arrows point to the smallest pair.
The black arrowheads point to the positively heteropycnotic telomeric regions. The white arrowheads mark the increase in separation of the
homologous chromosomes. Scale 5 10 mm.
224 THE JOURNAL OF ARACHNOLOGY

Figure 2.—DAPI staining of interphase nuclei and meiotic chromosomes of Brachistosternus alienus. a. Interphase; b. Leptotene-zygotene; c.
Late pachytene; d. Early postpachytene; e. Late postpachytene. Asterisks point to chromocenters. Arrows point to DAPI-positive bands showing
higher intensity of fluorescence. Arrowheads point to DAPI-positive bands exhibiting lower intensity of fluorescence. Scale 5 10 mm.

To determine the meiotic karyotype, chromosome measurements heterochromatin. Interphase and leptotene/zygotene nuclei usually
were performed in 11 well-spread postpachytene and prometaphase I contain one or two conspicuous DAPI-positive chromocenters as well
using Micro-Measure software, version 3.3 (Reeves & Tear 2000). The as some signals of intermediate or low intensity (Fig. 2a, b).
relative length of each bivalent was calculated as a percentage of total Pachytene karyotypes exhibit between eight and 10 intercalary
complement length (TCL; length of all bivalents of the karyotype). DAPI-positive bands showing higher intensity of fluorescence and
These data allowed us to construct an ideogram. Bivalents of seven some bands of lower intensity in terminal or intercalary regions
prometaphase I exhibiting the same degree of condensation were (Fig. 2c). During the following bivalent condensation, most of the
measured to determine absolute length (mm) of each bivalent. low-intensity bands become indistinguishable (Fig. 2d). At late
The spermatogonial mitosis shows 46 monocentric chromosomes; postpachytene, intercalary AT-rich bands can be identified in one
one pair is remarkable for its small size (Fig. 1a). During zygotene, large and four middle-sized bivalents, while terminal bands are
terminal regions of some bivalents exhibit small positively hetero- detected in two middle-sized bivalents (Fig. 2e).
pycnotic blocks (Fig. 1b), which are no longer distinguishable at Heterochromatin distribution has been analyzed in only a few
pachytene (Fig. 1c). Twenty-three bivalents are seen from late scorpions so far, namely eight species of Buthidae (belonging to
pachytene until prometaphase I (Fig. 1d–h). During the chromatin genera Isometroides, Isometrus, Lychas and Tityus) (Shanahan 1989a;
condensation, at early and middle postpachytene the homologous Schneider et al. 2009b; Schneider & Cella 2010), six species of
chromosomes of the bivalents remain tightly joined (Fig. 1d, e). At Urodacidae (genus Urodacus) (Shanahan 1989b), and two species of
late postpachytene it can be seen that the homologous chromosomes Bothriuridae belonging to the genus Bothriurus (Schneider et al.
lie parallel to each other, thus showing the absence of chiasmata, 2009a). Heterochromatin content and distribution in Buthidae is
which is a common feature of meiosis in male scorpions (Fig. 1f). At variable. In contrast to the other scorpions, this family shows
prometaphase I and metaphase I, many bivalents show a small region holokinetic chromosomes. Most heterochromatin of buthids occurs at
in the intercalary or terminal position where the homologues are more telomeric regions. Shanahan (1989b) reports pericentromeric bands in
separated (Fig. 1g–i). most Urodacus species studied. In Bothriurus rochensis Schneider et al.
The meiotic karyotype shows 23 homomorphic bivalents (Fig. 1j). (2009a) report small C-bands in the centromeric regions of some
Three groups of bivalents can be identified as follows: three large subtelocentric and submetacentric pairs, as well as in the terminal
bivalents of different size (4.58, 4.08 and 3.73 mm), 19 middle-sized region of the long arm of several submetacentric pairs. These authors
bivalents decreasing gradually in size (3.30, 3.13, 3.02, 2.92, 2.83, 2.73, also described AT-rich bands in the terminal regions of two pachytene
2.66, 2.59, 2.45, 2.38, 2.28, 2.16, 2.08, 1.95, 1.79, 1.76, 1.73, 1.57 and bivalents of B. rochensis. In contrast, heterochromatin has not been
1.44 mm), and the smallest bivalent of the complement (0.94 mm). The detected by C-banding and AT specific fluorochrome in Bothriurus
relative length of the bivalents ranges from 8.03% to 6.74% in the first araguayae (Schneider et al. 2009a). Blocks of presumed AT-rich
group, from 5.77% to 2.41% in the second group, and is 1.59% for the heterochromatin of Brachistosternus alienus may correspond to
smallest bivalent (Fig. 1k). pericentromeric regions of monobrachial and bibrachial chromo-
Fluorescent banding using DAPI revealed AT-rich regions, namely somes.
chromocenters at interphase nuclei as well as bands on bivalents at For Brachistosternus species evaluated so far, the haploid set of B.
prophase of the first meiotic division. These regions vary in size and ferrugineus and B. montanus is formed by 23 chromosomes. The same
number. They are presumably formed by AT-rich constitutive number is found in populations of B. pentheri from the northernmost
ADILARDI ET AL.—MEIOSIS OF BRACHISTOSTERNUS ALIENUS 225

limit of the species distribution (north of La Rioja province, Ferreira, A.P. 1968. Contribution to the knowledge of cytology of
Argentina) (Rodrı́guez Gil et al. 2009). Individuals of B. pentheri two species of Brazilian scorpions: Opisthacanthus manauarensis
from northern populations are larger and much less pigmented than Ferreira, 1967 (Scorpiones, Scorpionidae) and Bothriurus asper
the typical morph. On the other hand, populations corresponding to araguaiae (Scorpiones, Bothriuridae). Anais da Academia Brasi-
the holotypic morph, which are distributed from the south of La leira de Ciências 40:97–99.
Rioja province to the southeast of Buenos Aires province, have n 5 Giacomozzi, R.O. 1977. Citotaxonomı́a de Bothriuridae (Scorpio-
21 (Giacomozzi 1977; Rodrı́guez Gil et al. 2009). Rodrı́guez Gil et al. nida) de la Argentina. Physis (Buenos Aires) 37:236.
(2009) propose that marginal populations of B. pentheri from Lönnberg, E. 1898. On the scorpions obtained during the Swedish
northern Argentina could be considered a subspecies or even a expedition to Tierra del Fuego 1895. Svenska Expeditionen till
different species due to their specific morphological features and Magelländersna 2:45–48.
different chromosome number. Maury, E.A. 1972. Essai d’une classification des sous families de
The haploid karyotype of B. alienus from Comallo (province of Rı́o scorpions Bothriuridae. 5th International Congress of Arachnol-
Negro, Argentina) (this study) comprises 23 chromosomes, whereas ogy, Brno 1971:29–36.
the specimens of B. alienus studied by Giacomozzi (1977) have n 5 14. Mello-Leitão, C.F. de. 1934. Estudo monográfico dos escorpiões da
Giacomozzi (1977) stated that the specimens were determined by Dr. Republica Argentina. Octava Reunión de la Sociedad Argentina de
E. Maury and sampled in the province of Chubut, Argentina. Patologı́a Regional del Norte. Santiago del Estero 1933:1–97.
Although further information on the collection site is missing, data Mello-Leitão, C.F. de. 1945. Escorpiões Sul-Americanos. Arquivos
from that period suggests that the specimens were collected near do Museu Nacional 40:7–468.
Puerto Madryn, the species’ type locality. However, it is impossible to Ochoa, J.A. & A.A. Ojanguren-Affilastro. 2007. Systematics and
determine if the specimens studied by Giacomozzi (1977) belong to B. distribution of Brachistosternus (Brachistosternus) ehrenbergii
alienus or to B. angustimanus Ojanguren-Affilastro & Roig-Alsina (Gervais, 1841) with the first record of stridulation in this genus
2001 (a sympatric species), because the material is no longer available. Brachistosternus (Scorpiones. Bothriuridae). Studies on Neotrop-
At the time of Giacomozzi’s study, most authors based the ical Fauna and Environment 41:79–85.
identification of B. alienus on a redescription of this species by Ojanguren-Affilastro, A.A. 2001. Sistemática y distribución de
Mello-Leitão (1934, 1945), which corresponds more closely to B. Brachistosternus alienus Lönnberg (Scorpiones, Bothriuridae).
angustimanus than to the original description of B. alienus by Revista del Museo Argentino de Ciencias Naturales 3:169–174.
Lönnberg (1898). Both species are sympatric over most of their Ojanguren-Affilastro, A.A. 2003a. The genus Brachistosternus in
geographic range. Brachistosternus angustimanus is larger and usually Argentina, with the description of a new species from Patagonia
more abundant than B. alienus (Ojanguren-Affilastro 2001; Ojangu- (Scorpiones, Bothriuridae). Journal of Arachnology 31:317–331.
ren-Affilastro & Roig-Alsina 2001; Rodrı́guez Gil et al. 2009). Ojanguren-Affilastro, A.A. 2003b. Las especies andinas de Brachistos-
Conclusive evidence of species misidentification was provided by ternus (Leptosternus), con la descripción de tres nuevas especies
Maury (1972), who assigned a hemispermatophore of B. angustimanus (Scorpiones, Bothriuridae). Revista Ibérica de Aracnologı́a 8:23–36.
to B. alienus. Ojanguren-Affilastro, A.A. 2005a. Notes on the genus Brachistos-
The considerations mentioned above and the reduced chromosome ternus (Scorpiones, Bothriuridae) in Chile, with the description of
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rule out the possibility that Giacomozzi’s specimens correspond to B. escorpiones de la República Argentina. Revista Ibérica de
alienus; in this case, species should be considered polytypic, like B. Aracnologı́a 11:75–241.
pentheri (Rodrı́guez Gil et al. 2009). The specimens analyzed in this Ojanguren-Affilastro, A.A. & A. Roig-Alsina. 2001. Brachistosternus
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Affilastro 2001, 2005b). At this time access to living material of Ojanguren-Affilastro, A.A. & C.L. Scioscia. 2007. New data about
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individuals unequivocally identified as B. alienus and B. angustimanus Ojanguren-Affilastro, A.A. & M.J. Ramı́rez. 2009. Phylogenetic
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ACKNOWLEDGMENTS
the description of two new species. American Museum Novitates
This work was supported by grants from the National Council of 3564:1–44.
Scientific and Technological Research (CONICET) (PIP 0342), Ojanguren-Affilastro, A.A., P. Agusto, J. Pizarro Araya & C.I.
National University of Buenos Aires (UBA) (Ex 0859) and ANPCyT Mattoni. 2007b. Two new scorpion species of genus Brachistos-
(PICT 2010-1665) to Dr. L. Poggio and L. Mola and by a MACN ternus (Scorpiones: Bothriuridae) from northern Chile. Zootaxa
grant for collections improvement to C. Scioscia and A. A. 1623:55–68.
Ojanguren-Affilastro. The authors wish to thank L. Compagnucci, Prendini, L. 2003. A new genus and species of bothriurid scorpion
J.J. Martı́nez and L. Piacentini for their help in fieldwork and H. from the Brandberg Masif, Namibia, with a reanalysis of
Dinapoli for maintaining the scorpions in captivity. We are also Bothriurid phylogeny and a discussion of the phylogenetic position
indebted to J. Bond and two anonymous reviewers, who made very of Lisposoma Lawrence. Systematic Entomology 28:149–172.
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