UNIVERSIDADE ESTADUAL PAULISTA

JLIO DE MESQUITA FILHO

INSTITUTO DE BIOCINCIAS - RIO CLARO

CINCIAS BIOLGICAS - NOTURNO

LEILA MIGUEL STAVALE

CARITIPO, COMPORTAMENTO DOS CROMOSSOMOS NA MEIOSE

E REGIO ORGANIZADORA DE NUCLOLO DE ARANHAS
PERTENCENTES S FAMLIAS OXYOPIDAE E THERIDIIDAE
(ARANEOMORPHAE, ENTELEGYNAE)

Rio Claro
2009
 LEILA MIGUEL STAVALE

CARITIPO, COMPORTAMENTO DOS CROMOSSOMOS NA MEIOSE

E REGIO ORGANIZADORA DE NUCLOLO DE ARANHAS
PERTENCENTES S FAMLIAS OXYOPIDAE E THERIDIIDAE
(ARANEOMORPHAE, ENTELEGYNAE)

Orientador: Marielle Cristina Schneider

Co-orientador: Doralice Maria Cella

Trabalho de Concluso de Curso apresentado

ao Instituto de Biocincias da Universidade
Estadual Paulista Jlio de Mesquita Filho -
Cmpus de Rio Claro, para obteno do grau
de Bacharel e Licenciado em Cincias
Biolgicas.

Rio Claro
2009
595.44 Stavale, Leila Miguel
S798c Caritipo, comportamento dos cromossomos na meiose e regio organizadora de
nuclolo de aranhas pertencentes s famlias Oxyopidae e Theridiidae (Araneomorphae,
Entelegynae) / Leila Miguel Stavale. - Rio Claro : [s.n.], 2009
59 f. : il., figs.

Trabalho de concluso de curso (Licenciatura e Bacharelado - Cincias Biolgicas) -

Universidade Estadual Paulista, Instituto de Biocincias de Rio Claro
Orientador: Marielle Cristina Schneider
Co-Orientador: Doralice Maria Cella

1. Aracndeos. 2. Citogentica. 3. Evoluo. 4. Nmero diplide. 5. Quiasma. 6. Sistema

cromossmico sexual. I. Ttulo.

Ficha Catalogrfica elaborada pela STATI - Biblioteca da UNESP

Campus de Rio Claro/SP
Dedico este trabalho aos meus pais, Mrcia de

Lourdes Miguel Stvale e Maurcio Stvale, pelo amor

e apoio incondicionais.
 AGRADECIMENTOS

A Deus, por tudo que tem me proporcionado.

Aos meus pais, pelo amor, dedicao e apoio em todos os momentos.

minha famlia, pelo incentivo e carinho constantes.

s minhas orientadoras, Marielle Cristina Schneider e Doralice Maria Cella, pelos

ensinamentos, dedicao, pacincia e confiana que depositaram em mim.

Aos meus amigos, Hellen Maria Soares, Cynthia Renata de Oliveira Jacob e Wagner
Paschoal de Andrade Antonio, pela amizade, ajuda e risadas. Sentirei falta de
encontr-los todos os dias e das longas conversas.

s minhas amigas de Repblica, Jssica, Amanda(s), Marcinha e Keila, que se

tornaram irms para mim e por terem feito esses os melhores anos de minha vida.

Aos meus amigos de laboratrio, Emygdio de Paula Neto, Milena de Julio e Andr
M. Giroti, pela amizade e auxlio durante minha Iniciao Cientfica.

Ao meu namorado Rodrigo Luiz Mazzali Gallo, pelas palavras carinhosas e por estar
ao meu lado em todos os momentos.

A todos os colegas do curso de Cincias Biolgicas Noturno 2005, por terem

contribudo de alguma forma para meu crescimento pessoal e profissional.

A todos os professores que ofereceram oportunidades para eu conquistar meu

objetivo de ser biloga.

FAPESP (Fundao de Amparo Pesquisa do Estado de So Paulo) pela

concesso da bolsa de Iniciao Cientfica.

 A histria da Terra est gravada em sua

crosta, mas a histria de todos os organismos est

escrita em seus cromossomos.

KIHARA, 1975.
 SUMRIO

Pgina

1. RESUMO ............................................................................................................... 6

2. INTRODUO E REVISO BIBLIOGRFICA ..................................................... 8

2.1. CONSIDERAES GERAIS SOBRE ARANEAE ......................................................... 8

2.2. INFORMAES CITOGENTICAS EM ARANEAE ...................................................... 9

3. JUSTIFICATIVA E OBJETIVOS ............................................................................ 12

4. MATERIAL E MTODOS ...................................................................................... 13

4.1. MATERIAL ........................................................................................................... 13

4.2. MTODOS ........................................................................................................... 13

4.2.1. Obteno das preparaes cromossmicas ........................................... 13

4.2.2. Colorao convencional (Giemsa) .......................................................... 14

4.2.3. Impregnao pelo on prata .................................................................... 15

4.2.4. Colorao seqencial .............................................................................. 15

4.2.5. Anlises cromossmicas ......................................................................... 15

5. RESULTADOS E DISCUSSO ............................................................................. 16

6. REFERNCIAS BIBLIOGRFICAS ....................................................................... 53

 6

1. RESUMO
No presente trabalho, seis espcies de aranhas pertencentes s famlias
Oxyopidae e Theridiidae foram examinadas citogeneticamente, atravs de tcnicas
de colorao convencional e impregnao pelo on prata. A anlise de clulas
mitticas e meiticas coradas com Giemsa de quatro espcies de Oxyopidae,
Hamataliwa sp., Peucetia flava, Peucetia rubrolineata e Oxyopes salticus revelou
informaes citogenticas inditas para a famlia. Metfases espermatogoniais de
Hamataliwa sp. mostraram o caritipo 2n=26+X1X2, o qual corresponde ao maior
nmero diplide j descrito para a famlia. Clulas mitticas de P. flava e P.
rubrolineata exibiram 2n=20+X1X2 e 2n=20+X, respectivamente, indicando a
ocorrncia de uma variabilidade cariotpica dentro desse gnero. Os cromossomos
dessas trs espcies apresentaram morfologia acro/telocntrica. Os resultados
obtidos em O. salticus foram surpreendentes, pois revelaram 2n=10+X, o menor
nmero cromossmico encontrado para Oxyopidae e o segundo menor registrado
para aranhas do grupo Entelegynae, bem como morfologia meta/submetacntrica da
maioria dos cromossomos. Alm disso, um indivduo da amostra de O. salticus
examinada apresentou um heteromorfismo nos elementos que constituem o primeiro
par do caritipo e um cromossomo B em algumas clulas. Em Hamataliwa sp. e O.
salticus, as regies organizadoras de nuclolo estavam localizadas sobre dois e trs
pares autossmicos, respectivamente. Em relao s duas espcies de Theridiidae,
Argyrodes elevatus apresentou um caritipo totalmente discrepante, quando
comparado com aqueles j descritos para a famlia, uma vez que mostrou o nmero
diplide 2n=21, sistema cromossmico sexual do tipo X/XX e morfologia
cromossmica meta/submetacntrica. No entanto, as caractersticas cariotpicas
verificadas na maioria dos exemplares de Nesticodes rufipes foram semelhantes
aquelas mais freqentes em aranhas Theridiidae, ou seja, nmero diplide 2n=22,
 7

incluindo o sistema cromossmico sexual do tipo X1X2/X1X1X2X2 e cromossomos

com morfologia subtelo/acrocntrica. Alm disso, um exemplar macho apresentou
2n=24, devido a presena de um par adicional de cromossomos autossmicos, o
qual exibiu um comportamento regular durante a meiose. Clulas mitticas
impregnadas pelo on prata revelaram RONs sobre a regio terminal do brao curto
dos pares 2, 3 e 4 em A. elevatus e regio terminal do brao longo do par 4 em N.
rufipes. Atravs dos resultados obtidos no presente estudo, dos dados citogenticos
existentes na literatura e das hipteses filogenticas propostas para aranhas
Oxyopidae e Theridiidae foi possvel traar os principais mecanismos de evoluo
cromossmica que tem ocorrido em espcies dessas duas famlias.

Palavras-chave: citogentica, evoluo, nmero diplide, quiasma, sistema

cromossmico sexual
 8

2. INTRODUO E REVISO BIBLIOGRFICA

2.1. CONSIDERAES GERAIS SOBRE ARANEAE

A ordem Araneae o segundo txon mais diversificado da classe Arachnida,
incluindo 40.998 espcies (Platnick, 2009). No entanto, estimativas recentes indicam
que apenas um tero do total de espcies de aranhas existentes conhecido
atualmente (Brescovit, 1999). As aranhas esto divididas em trs grandes grupos
monofilticos, Mesothelae, Mygalomorphae e Araneomorphae (Coddington e Levi,
1991).
De acordo com caracteres morfolgicos, a subordem Mesothelae ocupa uma
posio basal na ordem Araneae (Coddington e Levi, 1991). Essa subordem
constituda por somente 87 espcies pertencentes a uma nica famlia, Liphistiidae,
e apresenta uma distribuio restrita s regies Oriental e Palertica (Coddington e
Levi, 1991; Platnick, 2009). A subordem Opisthothelae engloba as duas infra-ordens
Mygalomorphae e Araneomorphae, as quais formam um grupo-irmo. As
Mygalomorphae, aranhas conhecidas popularmente como caranguejeiras ou
tarntulas, possuem aproximadamente 1.400 espcies, distribudas nas regies
Australiana, Neotropical e Paleotropical. As Araneomorphae, freqentemente
denominadas de aranhas-verdadeiras, incluem 90% das espcies descritas e
ocorrem em quase todas as regies biogeogrficas (Coddington e Levi, 1991;
Brescovit, 1999; Platnick, 2009). A grande maioria dos representantes dessa ltima
infra-ordem est subdividida nos clados Haplogynae e Entelegynae. As haploginas
so Araneomorphae basais e esto representadas por 2.665 espcies, enquanto as
enteleginas, o grupo mais derivado, retm o maior nmero de aranhas conhecidas,
cerca de 38.300 espcies pertencentes a 73 famlias (Coddington e Levi, 1991;
Platnick, 2009).
 9

2.2. INFORMAES CITOGENTICAS EM ARANEAE

Citogeneticamente, 644 espcies de aranhas foram analisadas (Arajo, 2007,
Arajo et al., 2008; Krl, 2007; Oliveira et al., 2007; Rodriguez-Gil et al., 2007);
porm, esse nmero ainda pequeno e corresponde a menos que 2% das espcies
descritas sob o ponto de vista taxonmico. As informaes cariotpicas revelam que
em Araneae, o nmero diplide bastante diversificado, variando entre 2n=7 a
2n=96 (Suzuki, 1952, 1954). No entanto, o sistema cromossmico sexual do tipo
X1X2 nos machos o mais freqente, ocorrendo em 77% das espcies examinadas
at o presente momento (Arajo et al., 2005), e a morfologia acrocntrica dos
cromossomos predominante (Rowell, 1990).
O maior nmero cromossmico j descrito para uma aranha, 2n=96, foi para
um representante da subordem basal Mesothelae. Adicionalmente, o sistema
cromossmico sexual X1X2 foi registrado neste grupo (Suzuki, 1954). Considerando
essas caractersticas, a evoluo cariotpica das aranhas parece ter ocorrido atravs
da reduo do nmero diplide, manuteno do sistema cromossmico sexual X1X2
ou alterao para outros sistemas do tipo simples, como o XY e X, ou do tipo
mltiplo, como o X1X2X3 e X1X2X3X4 (Suzuki, 1954; Krl et al., 2006; Krl, 2007).
Cromossomos com morfologia meta/submetacntrica so comumente encontrados
em clados basais da ordem Araneae e ocorrem em espcies de Mygalomorphae
(ez et al., 2006), o que indica que este pode ser um carter ancestral do
caritipo das aranhas (Krl et al., 2006).
Entelegynae o grupo mais amplamente estudado com relao s
caractersticas cromossmicas, visto que existem dados de 578 espcies includas
em 37 famlias diferentes (Arajo, 2007). Nas enteleginas, o nmero diplide mais
baixo, 2n=10, foi descrito para um representante da famlia Uloboridae (Parida e
Sharma, 1987), enquanto que o mais alto, 2n=52, foi registrado para uma espcie
de Agelenidae (Wallace, 1909). Embora ocorra essa variao do nmero
cromossmico, Krl et al. (2006) sugeriram que o caritipo 2n=42=40+X1X2, com
cromossomos acrocntricos, poderia ser representativo de um estado ancestral para
as aranhas enteleginas. Essa hiptese foi baseada no fato que a frmula cariotpica
acima mencionada est presente em diversas famlias no relacionadas de
Entelegynae bem como em clados basais desse grupo. No entanto, anlises
 10

citogenticas adicionais, utilizando tcnicas de colorao cromossmica

convencional e diferencial, precisam ser realizadas nas enteleginas para confirmar
essa hiptese e entender os processos de evoluo cromossmica que tm ocorrido
em cada famlia.
Dentro de Entelegynae encontram-se as famlias Oxyopidae e Theridiidae, as
quais so derivadas e pertencem s superfamlias Lycosoidea e Araneoidea,
respectivamente (Coddington e Levi, 1991).
Lycosoidea possui 149 espcies das famlias Ctenidae, Lycosidae, Miturgidae,
Oxyopidae, Pisauridae, Psechridae e Zoridae examinadas citogeneticamente
(Arajo, 2007). Em representantes de todas essas sete famlias, com exceo de
Oxyopidae, o caritipo 2n=28=26+X1X2, com cromossomos acrocntricos,
prevalecente. Em contraste, na famlia Oxyopidae, o nmero diplide 2n=21 e o
sistema cromossmico sexual do tipo X foram observados na maioria das espcies.
Porm, outras frmulas cariotpicas tambm ocorrem nesse txon como, por
exemplo, 2n=22=20+X1X2 em Oxyopes salticus, 2n=23=22+X em Oxyopes
javanus e Oxyopes macilentus, e 2n=28=26+X1X2 em Peucetia viridiana. Com
relao morfologia cromossmica, somente 11 espcies foram caracterizadas, as
quais mostraram cromossomos principalmente do tipo acro/telocntricos (Arajo,
2007).
A superfamlia Araneoidea um dos grupos mais conhecidos citogeneticamente,
com dados de 185 espcies distribudas nas famlias Araneidae, Linyphiidae,
Nephilidae, Nesticidae, Theridiidae e Tetragnathidae (Arajo, 2007). Nas espcies
dessa superfamlia, a frmula cariotpica 2n=24=22+X1X2 bastante conservada,
sendo predominante em todas as famlias, exceto em Theridiidae. As informaes
citogenticas existentes para Theridiidae mostraram que o caritipo
2n=22=20+X1X2 compartilhado por 23 espcies dentre as 29 j analisadas,
ocorrendo em 11 gneros diferentes, com exceo de Chrysso e Latrodectus
(Arajo, 2007). Os quatro representantes de Latrodectus estudados revelaram uma
grande diversidade cariotpica, com nmero diplide variando entre 2n=16 a
2n=28, os quais representam, respectivamente, os mais baixos e altos nmeros
cromossmicos verificados para a famlia. De modo similar ao que ocorre em outras
 11

aranhas Entelegynae, todas as 16 espcies de Theridiidae, cujos cromossomos

foram classificados, exibiram invariavelmente morfologia acro/telocntrica.
Os cromossomos de todas as espcies das famlias Oxyopidae e Theridiidae
foram analisados principalmente com tcnicas de colorao convencional, existindo
um nico relato sobre a ocorrncia de regio organizadora de nuclolo (RON) sobre
um par autossmico em uma espcie de Oxyopidae (Barrion et al., 1989). A
identificao das regies organizadoras de nuclolo (RONs) de grande importncia
para comparaes cariotpicas de espcies relacionadas e em estudos evolutivos
(Petitpierre, 1996). Adicionalmente, a determinao do nmero e localizao das
RONs constitui um critrio adicional na caracterizao cromossmica das espcies,
pois essas regies esto sempre presentes nos cromossomos e podem apresentar
um padro de distribuio conservado evolutivamente para um determinado grupo
de espcies. Alteraes na distribuio das RONs podem ser indicativas de
ocorrncia de rearranjos cromossmicos e fornecer dados sobre os processos de
diferenciao cariotpica entre espcies relacionadas (Oliveira, 2004). Em algumas
poucas espcies de aranhas cujo padro de RON foi estabelecido,
aproximadamente 15, verifica-se que essa regio pode estar localizada sobre os
cromossomos sexuais e autossomos em representantes de Haplogynae e em pares
autossmicos de Entelegynae (Wise, 1983; Krl et al., 2006; Oliveira et al., 2007;
Rodriguez-Gil et al., 2007; Arajo et al., 2008).
 12

3. JUSTIFICATIVA E OBJETIVOS
Considerando as particularidades cromossmicas encontradas em espcies das
famlias Oxyopidae e Theridiidae, cujos cromossomos foram predominantemente
investigados com tcnicas de colorao convencional, o fato de apenas 5% das
aranhas Oxyopidae e 1.5% dos representantes de Theridiidae terem sido analisados
sob o ponto de vista citogentico e de no existirem registros cariotpicos para
representantes da fauna brasileira, o presente estudo tem como objetivos
caracterizar citogeneticamente seis espcies de aranhas Oxyopidae e Theridiidae,
visando traar os principais mecanismos de evoluo cariotpica. Para tal, foi
determinado:
- o nmero e a morfologia dos cromossomos, o tipo de sistema cromossmico
sexual, e o comportamento dos cromossomos durante a meiose quanto
heteropicnose, nmero e localizao de quiasmas, configurao dos cromossomos
sexuais e segregao anafsica;
- o padro de distribuio das RONs.
 13

4. MATERIAL E MTODOS

4.1. MATERIAL
Neste trabalho, uma amostra de 85 indivduos da fauna brasileira foi analisada,
sendo 27 exemplares pertencentes famlia Oxyopidae e 58 famlia Theridiidae
(Tabela 1). Os espcimes foram identificados taxonomicamente pelo Dr. Antonio
Domingos Brescovit, do Laboratrio de Artrpodes, Instituto Butantan, So Paulo,
SP, Brasil.

Tabela 1 Espcies das famlias Oxyopidae e Theridiidae, com seus respectivos nmero de
exemplares analisados neste trabalho e locais de coleta.
Espcies Adultos Embries Procedncia
Oxyopidae
Hamataliwa sp 2 e 1 ----- Rio Claro (2224S, 4733W), SP
Peucetia flava 1 e 3 ----- Rio Claro (2224S, 4733W), SP
Peucetia rubrolineata 9 ----- Rio Claro (2224S, 4733W), SP
Oxyopes salticus 7 e 4 ----- Rio Claro (2224S, 4733W), SP
Theridiidae
Argyrodes elevatus 13 12 e 11 Rio Claro (2224S, 4733W), SP e Tup (2156S, 5030W), SP
Nesticodes rufipes 6 e 7 6 e 3 Rio Claro (2224S, 4733W), SP e Viosa (2045'S 4252'W), MG

4.2. MTODOS
4.2.1. Obteno das preparaes cromossmicas
As preparaes citolgicas, para o estudo dos cromossomos mitticos e
meiticos, foram obtidas de testculos e/ou ovrios de indivduos adultos, conforme
descrito abaixo:
a) dissecar o animal em soluo fisiolgica para insetos (128.3 mM de NaCl, 16.7
mM de Na2HPO4, 19.9 mM de KH2PO4), retirar as gnadas e transferi-las para
 14

um recipiente contendo colchicina 0.16% (em soluo fisiolgica para insetos),

deixando por duas horas;
b) acrescentar um volume de gua de torneira igual ao de colchicina e deixar por 15
minutos;
c) adicionar 10 gotas de fixador Carnoy I (metanol e cido actico 3:1) e deixar
por 1 minuto;
d) transferir as gnadas para um recipiente contendo fixador Carnoy I e deixar por
30 minutos, no mnimo;
e) macerar, sobre uma lmina, uma das gnadas, juntamente com uma gota de
cido actico 45%;
f) secar a lmina em uma placa de metal temperatura de 35 a 40C.

Alternativamente, algumas preparaes cromossmicas foram obtidas a partir de

clulas embrionrias, de acordo com a tcnica descrita a seguir:
a) dissecar o ovo em soluo fisiolgica para insetos, remover o embrio e
transferi-lo para uma placa escavada contendo colchicina 0.05% (em soluo
fisiolgica para insetos), deixando por 2 horas;
b) adicionar um volume de gua de torneira igual ao volume de colchicina e deixar
durante 15 minutos;
c) remover cuidadosamente toda a soluo (colchicina e gua de torneira) e fixar o
material em Carnoy I por 1 minuto. Esse processo de fixao deve ser repetido
por trs vezes, sendo que na ltima fixao o embrio deve ser mantido por 30
minutos, no mnimo;
d) transferir o embrio para uma lmina e macer-lo, com o auxlio de um basto
de metal, em algumas gotas de cido actico 60%;
e) secar a lmina em uma placa de metal, temperatura de 35 a 40C.

4.2.2. Colorao convencional (Giemsa)

a) corar a lmina com soluo de Giemsa a 3% (47mL de gua destilada, 1.5mL de
soluo comercial de Giemsa e 1.5mL de tampo fosfato pH 6.8) durante 15
minutos;
b) lavar a lmina em gua destilada e sec-la ao ar.
 15

4.2.3. Impregnao pelo on prata para deteco das RONs, segundo Howell e
Black (1980)
a) colocar sobre a lmina uma gota de soluo coloidal reveladora (1g de gelatina
Merck dissolvida em 50 mL de gua destilada + 0.5 mL de cido frmico) duas
gotas de soluo de nitrato de prata 50% e cobrir com lamnula;
b) incubar a lmina em cmara mida, durante 3-5 minutos, temperatura de 67C;
c) remover a lamnula com um jato de gua destilada, lavar a lmina e secar ao ar.

4.2.4. Colorao seqencial

Algumas preparaes cromossmicas foram analisadas com colorao
seqencial, a qual consiste em submeter os cromossomos tcnica de colorao
convencional e posteriormente, tcnica de impregnao pelo on prata.

4.2.3. Anlises cromossmicas

As anlises dos cromossomos foram realizadas em microscopia de luz. As
clulas mitticas e meiticas foram capturadas em um fotomicroscpio Olympus
BX51, com objetiva 100x de imerso, optovar 1.6, utilizando o software DP
Controller. A morfologia dos cromossomos foi determinada de acordo com a
nomenclatura proposta por Levan et al. (1964).
 16

5. RESULTADOS E DISCUSSO

Os resultados obtidos atravs da anlise citogentica de quatro espcies de

Oxyopidae e duas de Theridiidae so apresentados na forma de dois artigos
cientficos.
 17

Chromossomal characteristics and karyotype evolution of Oxyopidae spiders

(Araneae, Entelegynae)

Leila Miguel Stavale1, Marielle Cristina Schneider1, Antonio Domingos Brescovit2,

Doralice Maria Cella1

1
Departamento de Biologia, UNESP, Rio Claro, So Paulo, Brazil
2
Laboratrio de Artrpodes, Instituto Butantan, So Paulo, So Paulo, Brazil

Key words: Hamataliwa, Peucetia flava, Peucetia rubrolineata, Oxyopes salticus,

meiosis, nucleolar organizing region, sex chromosome system
 18

Introduction
The spiders constitute one of the most diverse orders of animals and are
certainly the most abundant terrestrial predators (Coddington & Levi 1991).
Cytogenetically, only about 1.5% (Krl et al. 2006) among the 40.998 species
belonging to the order Araneae (Platnick 2009) were analyzed, which showed a wide
diversity of diploid chromosome number, ranging from 2n=7 to 2n=96, and types
of sex chromosome system, e.g., XY, X, X1X2, X1X2Y, X1X2X3, X1X2X3X4 (Arajo
2007). In species of the monophyletic basal groups, the highest chromosome
numbers were verified, such as 2n=96 in Mesothelae and 2n=86 in
Mygalomorphae (Suzuki 1954; Srivastava & Shukla 1986). Among the derivative
groups included in the infraorder Araneomorphae, the spiders of the Haplogynae
lineage present a predominance of low diploid chromosome numbers that vary
between 2n=7 and 2n=37, sex chromosome system of the X type, and
meta/submetacentric chromosomes. These features contrast with those of the sister-
group Entelegynae that possesses the highest number of species taxonomically
described and cytogenetically investigated (Arajo 2007; Platnick 2009). In
entelegyne spiders there is conservation of the diploid number 2n=42, sex
chromosome system of the X1X2 type, and chromosomes with acro/telocentric
morphology (Arajo et al. 2005; Krl et al. 2006).
After comparing the karyotype characteristics of basal and derived Araneae
species, Suzuki (1954) proposed that chromosomal evolution in this order has
occurred through reduction in the chromosome number. Later, Rowell (1990)
suggested that in spiders there is a peculiar form of karyotype evolution via "all or
nothing" fusions. This proposition was based on the fact that intermediate karyotypes
that include both acro/telocentric chromosomes and meta/submetacentric fused
chromosomes are rarely encountered in this group. Recently, Krl et al. (2006)
verified that certain related species of entelegyne differ with regard the diploid
number but no in relation to chromosome morphology that is maintained as
acro/telocentric. Thus, the authors attributed the reduction in the chromosome
number in these spiders to tandem fusions.
Within Entelegynae lineage, the family Oxyopidae is very interesting because of
its predominance of the 2n=22+X. This karyotype includes a diploid chromosome
 19

number that is relatively lower when compared with those of other families of this
group and a sex chromosome system that was observed in only 12% of the
entelegyne spiders. The chromosome morphology, however, described for the 11
species among a total of 21 studied is conserved as acro/telocentric (Table 1).
Oxyopidae belongs to the group of spiders known as true lycosoids, whose
monophyly is well-supported by morphological and molecular characters (Silva
Davila 2003). The families that constitute this group form the clade (((Psechridae
(Oxyopidae + Senoculidae)) + ((Trechaleidae (Lycosidae + Pisauridae))). In order to
increase the knowledge about the chromosomal characteristics and understand the
processes of karyotype evolution of Oxyopidae, four species belonging to three
different genera were cytogenetically analyzed with standard staining and silver
impregnation. It is worth emphasizing that this is the first chromosome record of
oxyopids from Brazilian fauna.
 20

Table 1 Oxyopidae species cytogenetically analyzed. A=acrocentric; M=metacentric; SM=submetacentric; T=telocentric; H=holocentric
Species Chromosomal Chromosomal Collection Reference
formula (2n males) morphology locality
Hamataliwa sp. 28=26+X1X2 26T+X1X2T Brazil Present work
Nishina generosa Komatsu (nonem nudum) 21=20+X 20A+XA Japan Suzuki (1952)
Oxyopes hindostanicus Pocock, 1901 21=20+X 20A+XA India Bole-Gowda (1950), Mittal (1961, 1970)
Oxyopes javanus Thorell, 1887 23=22+X 22H+XH Philippines Barrion et al. (1989)
Oxyopes lepidus (Blackwall, 1864) 21=20+X 20A+XA India Bole-Gowda (1958)
under Oxyopes similaris
Oxyopes macilentus L. Koch, 1878 21=20+X 20T+XT Taiwan Chen (1999)
23=22+X 22T+XT
Oxyopes pandae Tikader, 1969 21=20+X ----- India Srivastava & Shukla (1986)
Oxyopes ramosus (Martini & Goeze, 1778) 21=20+X 20A+XM Finland Hackman (1948)
Oxyopes ratnae Tikader, 1970 21=20+X 20T+XT India Datta & Chatterjee (1983, 1989), Parida & Sharma (1987),
Sharma & Parida (1987)
Oxyopes rufisternis Pocock, 1901 21=20+X 20A+XA India Mittal (1961, 1970)
Oxyopes ryvesi Pocock, 1901 21=20+X ----- India Sharma et al. (1960)
Oxyopes salticus Hentz, 1845 22=20+X1X2 ----- ----- Painter (1914)
Oxyopes salticus Hentz, 1845 11=10+X 6M+2SM+2A+XSM Brazil Present work
12=11+X/ 5M+2SM+4A+XSM/
13=11+X+B 5M+2SM+4A+XSM+BSM
Oxyopes scalaris Hentz, 1845 21=20+X ----- United States Tugmon et al. (1990)
Oxyopes sertatus L. Kock, 1877 21=20+X 20A/T+XA/T Japan, Taiwan Suzuki (1950, 1952), Igarashi & Kondo (1977), Chen (1999)
Oxyopes shweta Tikader, 1970 21=20+X ----- India Parida & Sharma (1987), Sharma & Parida (1987)
Oxyopes sushilae Tikader, 1965 21=20+X ----- India Srivastava & Shukla (1986)
Oxyopes sp 21=20+X ----- India Sharma et al. (1960)
Oxyopes sp. 22=20+X1X2 ----- India Mittal (1961)
Oxyopes sp. 22=20+X1X2 20A+X1X2A India Mittal (1970)
Oxyopes sp. 21=20+X ----- India Srivastava & Shukla (1986)
Oxyopes sp. 21=20+X ----- India Srivastava & Shukla (1986)
Peucetia flava Keyserling, 1877 22=20+X1X2 20T+X1X2T Brazil Present work
Peucetia rubrolineata Keyserling, 1877 21=20+X 20T+XT Brazil Present work
Peucetia viridana (Stoliczka, 1869) 28=26+X1X2 26A+X1X2A India Bole-Gowda (1950), Parida & Sharma (1987), Sharma &
Parida (1987)
 21

Materials and Methods

In this work, a sample of 27 individuals was analyzed, which included: two males
and one female of Hamataliwa sp., one male and three females of Peucetia flava
Keyserling, 1877, nine males of Peucetia rubrolineata Keyserling, 1877, and seven
males and four females of Oxyopes salticus Hentz, 1845. All individuals were
collected in Rio Claro (2224'S, 4733'W), state of So Paulo (SP), Brazil. The
voucher specimens were deposited in the Laboratrio de Artrpodes, Instituto
Butantan (IBSP), So Paulo, SP, Brazil. The chromosomal preparations were
obtained according to the procedure by Araujo et al. (2008). Chromosome spreads
were stained with Giemsa solution (3% of commercial Giemsa and 3% of phosphate
buffer, pH 6.8, in distilled water) for 15 min and subsequently silver-impregnated
(Howell & Black 1980) to detect the nucleolar organizer regions (NORs). Mitotic and
meiotic nuclei records were performed using an Olympus BX51 microscope. The
morphology of the chromosomes was determined according to the nomenclature
proposed by Levan et al. (1964).

Results
Mitotic metaphase cells of Hamataliwa sp. stained with Giemsa showed the
diploid numbers 2n=28 for males and 2n=30 for female, consistent with a
X1X2/X1X1X2X2 sex chromosome system (Figure 1A). All chromosomes revealed
acro/telocentric morphology and decreased gradually in size. The sex chromosomes
presented a high degree of condensation; the X1 sex chromosome was slightly larger
than the X2 chromosome, but both chromosomes possessed medium size. Early
prophase I cells of male Hamataliwa sp. revealed two blocks highly condensed and
positively heteropycnotic, which were interpreted as sex chromosomes (Figure 1B).
Diplotene and diakinesis nuclei showed 13II+X1X2 (Figure 1C-D), confirming the
diploid number and type of sex chromosome system established through analyses of
mitotic cells. In these meiosis-phases, the autosomal bivalents showed one interstitial
or terminal chiasma and the X1 and X2 chromosomes always appeared as univalents
and arranged side by side. In metaphase II cells, the haploid sets with n=13+X1X2
and n=13 chromosomes were observed (Figure 1E-F). All chromosome preparations
 22

of Hamataliwa sp. were subjected to silver impregnation but only diplotene and
diakinesis nuclei revealed NORs, which were located on the terminal region of one
medium-sized bivalent and interstitial region of one smallsized bivalent (Figure 1G-
H).
Giemsa-stained spermatogonial metaphases of P. flava and P. rubrolineata
demonstrated the diploid chromosome numbers 2n=22 and 2n=21, respectively. The
former species presented a sex chromosome system of the X1X2 type while the latter
showed a system of the X type (Figure 2A-B). The karyotypes of both species were
composed of acro/telocentric chromosomes that gradually varied in size and sex
chromosomes with similar size to smallest elements of the diploid complement. In
pachytene cells of P. flava and P. rubrolineata, the autosomal bivalents were fully
synapsed and the sex chromosomes appeared as highly condensed and positively
heteropycnotic univalents (Figure 2C and G). In diplotene and diakinesis nuclei, the
meiotic formula 10II+X1X2 for P. flava and 10II+X for P. rubrolineata was observed
(Figure 2D, E, H and I). In both species, all autosomal bivalents showed one
interstitial or terminal chiasma, with the exception of some cells of P. flava that
presented one bivalent with two terminal and/or interstitial chiasmata. Metaphase II
cells exhibited n=10+X1X2 and n=10 in P. flava (Figure 2F) and n=10+X and n=10 in
P. rubrolineata (Figure 2J), confirming the regular segregation of all chromosomes
during preceding anaphase. Although the chromosome preparations of the two
Peucetia species have been silver-impregnated, the presence of an argentophilic
material corresponding to the NORs was not verified in the sample of cells examined.
Mitotic metaphase cells of six males O. salticus after standard stained with
Giemsa showed the diploid number 2n=11, sex chromosome system of the X type,
and chromosomes with metacentric (pairs 1, 3 and 4), submetacentric (pair 2 and X
chromosome) and acro/telocentric (pair 5) morphology (Figure 3A). In relation to the
size, the chromosomes could be classified in large (pair 1), medium (pairs 2-4 and X
chromosome) and small (pair 5). Spermatogonial cells of one individual of the sample
examined of O. salticus showed discrepant characteristics to those above mentioned
(Figure 3B), i.e., the karyotype was composed of 2n=12 chromosomes, including
three heteromorphic chromosomes (one metacentric of large size and two
acro/telocentric of different sizes), four pairs of homomorphic chromosomes that
 23

correspond to pairs 2 to 5, and one submetacentric element identified as X sex

chromosome. The three unpaired autosomes represented the heterozygous
condition for the centric fusion between the chromosomes that originated the largest
pair of the diploid complement. Moreover, in some cells of this specimen with 2n=12,
the presence of one extra chromosome possessing submetacentric morphology and
extremely small size was detected (Figure 3C). This element was interpreted as B
chromosome. Thus, the spermatogonial cells of this individual exhibited 2n=12=11+X
or 2n=13=11+X+B. Description of male meiosis of O. salticus was only based on the
individuals that presented 2n=11 chromosomes. Pachytene nuclei exhibited 10
double filaments and one positively heteropycnotic block that was representative of
the X chromosome (Figure 3D). Diplotene cells revealed the meiotic formula 5II+X.
Most autosomal bivalents presented a variable number of interstitial and/or terminal
chiasmata from two to four, except the smallest bivalent that invariably showed one
interstitial or terminal chiasma (Figure 3E). During the prophase I, the X sex
chromosome was easily identified due to its high degree of condensation in relation
to the autosomes. Metaphase II spermatocytes showed two different haploid
numbers n=5+X and n=5 (Figure 3F-G). Silver-impregnated spermatogonial
metaphases revealed six NORs on the long arm terminal region of pairs 2 and 5 and
short arm terminal region of pair 3. However, only up to four NORs per cell were
active (Figure 3H-K).
 24

Figure 1 - Testicular cells of Hamataliwa sp. Giemsa-stained (A-G) and silver-impregnated (H). A.
Mitotic metaphase with 2n=26+X1X2 and telocentric chromosomes. B. Pachytene, showing positively
heteropycnotic sex chromosomes. C-D. Diplotene and diakinesis, exhibiting one interstitial (large
arrow) or terminal (small arrow) chiasma per bivalent. E-F. Metaphase II nuclei with n=13+X1X2 and
n=13, respectively. G. Diakinesis. H. The same cell as in G, revealing NORs (arrowhead) on terminal
and interstitial regions of two bivalents. In detail, bivalents with one terminal and interstitial NORs.
Scale=10m.
 25

Figure 2 - Testicular cells of Peucetia flava (A, C-F) and Peucetia rubrolineata (B, G-J) stained with
Giemsa. A and B. Karyotypes, showing 2n=20+X1X2 and 2n=20+X, respectively. Note the telocentric
morphology of all chromosomes. C and G. Pachytene with completely synapsed autosomal bivalents
and highly condensed and stained sex chromosomes. D and H. Diplotene. E and I. Diakinesis. Large
arrows point to interstitial chiasma and small arrows indicate terminal chiasma. F and J. Two cells in
late metaphase II whose sister-chromatids are separated. Scale=10m.
 26

Figure 3 - Testicular cells of Oxyopes salticus after staining with Giemsa (A-H, J) and silver
impregnation (I and K). A. Karyotype with 2n=10+X and metacentric, submetacentric, and acrocentric
chromosomes. B. Karyotype with 2n=11+X. Note the heterozygous condition for the centric fusion
between chromosomes that constitute the pair 1. In detail, the heteromorphic pair 1. C. Mitotic
metaphase, 2n=11+X+B, revealing the presence of one B chromosome. In detail, the submetacentric
B chromosome. D. Pachytene. E. Diplotene, 2n=5II+X, showing autosomal bivalents with interstitial
(large arrow) and/or terminal (small arrow) chiasmata. In details, bivalents with three and four
chiasmata. F-G. Metaphase II cells with n=5+X and n=5, respectively. H. Mitotic metaphase, 2n=10+X.
I. The same cells as in H, revealing NORs (arrowhead) on the terminal region of pairs 2, 3 and 5. J.
Incomplete mitotic metaphase with 2n=10. K. The same cells as in J, exhibiting NORs (arrowhead) on
the terminal region of pairs 2 and 3. Scale=10m.
 27

Discussion
The family Oxyopidae possesses 425 species subdivided into nine genera
(Platnick 2009). Until now, a total of 21 species of the genera Nishina, Oxyopes, and
Peucetia were cytogenetically investigated. Among the four oxyopids studied here,
only O. salticus have previously been examined by Painter (1914). Additionally, this
is the first chromosome record for a representative of the Hamataliwa genus. The
2n=26+X1X2 with acro/telocentric chromosomes observed in Hamataliwa sp. was
similar to the karyotype found in only one other species of this family, Peucetia
viridana (Bole-Gowda 1950; Parida & Sharma 1987; Sharma & Parida 1987). It is
worth pointing out that 2n=28 represents the highest diploid number already
described for Oxyopidae. The karyotype characteristics verified in the two Peucetia
species, 2n=20+X1X2 in P. flava and 2n=20+X in P. rubrolineata, as well as the
karyotype registered for P. viridana, 2n=26+X1X2, revealed that in this genus there
is a heterogeneity with regard the diploid number and type of sex chromosome
system. The acro/telocentric chromosomal morphology encountered in the three
species here investigated is however, similar to that predominant in Oxyopidae
spiders (Table 1). In relation to the size of the chromosomes, the only data is referent
to the sex chromosome of three species of the genus Oxyopes, in which the X
chromosome can be the largest or smallest element of the diploid complement. This
interspecific discrepancy regarding the size of the X chromosome could be related to
variations in the quantity of constitutive heterochromatin and probably did not indicate
independent origin of this sex chromosome.
The results obtained in most individuals of the sample of O. salticus examined in
the present work were surprisingly due to the occurrence of the diploid chromosome
number 2n=11. This diploid number is the lowest already encountered in
Oxyopidae and is the second lowest registered for Entelegynae spiders as a whole.
The predominance of biarmed chromosomes is also very rare in entelegynes and
has been verified in only 14 non-related species among a total of approximately 590
analyzed about the cytogenetic point of view (Hackman 1948; Suzuki 1951, 1954;
Bole-Gowda 1952; Mittal 1966; Rowell 1985, 1988, 1990, 1991; Sharma & Parida
1987; Hong et al. 1992; Amalin et al. 1993; Tsurusaki et al. 1993; Hancock & Rowell
1995; Krl 1995; Qingtao et al. 1996; Gorlova et al. 1997; Arajo 2007). In addition,
 28

the chromosome characteristics of O. salticus from the Rio Claro population differed
in relation to the karyotype already described for this same species, 2n=20+X1X2,
and for other 18 species of the Oxyopes genus that showed 2n=22+X and
2n=20+X (Table 1).
Within the Oxyopidae, the great diversity of diploid number and type of sex
chromosome system has probably origin from the karyotype 2n=26+X1X2 with
acro/telocentric chromosomes. This karyotype certainly constitutes the basal type for
this family, considering that it occurred in Senoculidae that is sister-group to
Oxyopidae and is commonly encountered in species of others families closely related
to Oxyopidae, such as Trechaleidae, Lycosidae, and Pisauridae (Arajo 2007; A. M.
Giroti, pers. comm. 2008). Therefore, chromosomal rearrangement of the tandem
fusion type between the autosomes was the mechanism responsible for the reduction
of the diploid number, and tandem fusion between the X1 and X2 chromosome give
rise to X sex chromosome system. Moreover, the karyotypes observed in the
individuals of O. salticus examined in this work revealed that centric fusions involving
the autosomes and the sex chromosomes also occurred during the karyotype
evolution of Oxyopidae spiders. Krl et al. (2006) analyzed certain species of
Haplogynae spiders and also proposed that the X sex chromosome system is
derived from a multiple sex chromosome system. In contrast with the hypothesis
formulated by us, i.e., that the X sex chromosome system arose by tandem or centric
fusion between the X1 and X2 of the X1X2 system, Krl et al. (2006) proposed that X
system has origin of the X1X2Y system. According to the authors, the conversion of
the X1X2Y into an X system was a gradual process and has the XY system as an
intermediate stage.
The unusual karyotype 2n=10+X, with a predominance of meta/submetacentric
chromosomes, encountered in the sample of O. salticus here studied is certainly a
derived type and evolved from 2n=20+X1X2 described by Painter (1914) through "all
or nothing" fusions as shows the Figure 4. We suggested that karyotype of O.
salticus from Rio Claro have origin by three main events: 1) centric fusion between
eight autosomal pairs, producing four pairs of meta/submetacentric chromosomes; 2)
centric fusion between the acro/telocentric X1 and X2 chromosomes, converting the
sex chromosome system into an X system; 3) tandem fusion between one ancestral
 29

autosomal pair of small size and the 1st derived autosomal pair, originating an
asymmetry in the derived karyotype due to the presence of one large and one small
autosomal pair. Furthermore, the sympatric occurrence of one individual of O.
salticus with the karyotype 2n=11+X, including the heterozygous state of the centric
fusion that originated the pair 1, revealed that the chromosome constitutions of O.
salticus with all chromosomes in a fused state is not totally established in the
population from Rio Claro. The chromosome characterization of a higher number of
specimens from population of Rio Claro and other localities is very interesting to be
realized and can reveal the presence of other karyotypes for this species.
Surprisingly, the diploid number variability in O. salticus was not only related to
the occurrence of autosomal heteromorphism but also due to the presence of B
chromosome. In spiders, the description of B chromosome is extremely sporadic and
was only registered for six species of the families Amaurobiidae, Clubionidae,
Lycosidae, Salticidae, Theridiidae, and Thomisidae (Montgomery 1905; Painter 1914;
Avills & Maddison 1991; Rowell & Main 1992; Qingtao et al. 1996). Unfortunately, in
the specimen of O. salticus carrier of the B chromosome, the meiotic cells presented
a low resolution due to chromosomal superposition, making it impossible to verify the
synaptic and segregational behavior of this additional chromosome.
The analysis of meiotic cells of Hamataliwa sp., P. flava, P. rubrolineata, and O.
salticus permitted us to confirm the diploid number, chromosomal morphology, and
mainly the type sex of chromosome system established through the study of mitotic
cells in the four species. Additionally, the investigation of diplotene and diakinesis
nuclei showed that one chiasma per bivalent that is frequently observed in other
oxyopids also occurred in Hamataliwa sp., P. flava, P. rubrolineata. In contrast, the
high number of chiasmata per bivalent was verified in all large-sized bivalents of O.
salticus. Similarly to this last species, the presence of more than one chiasma per
bivalent was also observed in some beetles whose diploid complement seems to be
evolved by fusions (Schneider et al. 2007). According to John (1990) the number of
chiasma per bivalent is partially dependent of the chromosome size. In O. salticus, a
clear relationship between the chromosome size and the number of chiasmata per
bivalent was noticed, since the small bivalent invariably exhibited only one interstitial
or terminal chiasma.
 30

The information about NOR in spiders is very scarcity. Within Entelegynae the
analysis of this specific chromosome region was performed only in one species of
Lycosidae, Nephillidae, Oxyopidae, and three species of Sparassidae, which
revealed NORs on terminal region of one, two, or up to three autosomal pairs (Wise
1983; Barrion et al. 1989; Arajo et al. 2005; Rodriguez-Gil et al. 2007). Although
Hamataliwa sp. and O. salticus differed regarding the number of carrier pairs of NOR,
these two species always presented argentophilic material on autosomal
chromosomes. The occurrence of NOR only on autosomal chromosomes could be a
shared characteristic to Entelegynae spiders, differing from Haplogynae, in which
Oliveira et al. (2007) proposed that NORs on both autosomes and sex chromosomes
seems to be the ancestral pattern for the group.
The analysis of the four Oxyopidae spiders and available data of the literature
permitted us to suggest the trends of chromosome evolution for this family. Despite
certain species to retain the ancestral chromosome constitutions 2n=26+X1X2 with
acro/telocentric chromosomes, the vast majority of the oxyopids have their karyotype
differentiated by both reduction in diploid chromosome number and change of the
sex chromosome system to X type. These mechanisms of chromosome evolution
resulted in an interspecific and intraspecific karyotype variability within the family
Oxyopidae. The most remarkable karyotype differentiation occurred in the specimens
of O. salticus studied here that showed one of the lowest diploid number already
recorded for Entelegynae spiders. The use of techniques to highlight specific
chromosome regions in a high number of species certainly will be very useful to
reveal more detail of the chromosome evolution in oxyopids.
 31

Figure 4 Schematic drawing, showing a probably origin of the different chromosome constitutions
observed in Oxyopes salticus. A. Ancestral karyotype with 2n=20+X1X2 and acro/telocentric
chromosomes. B. Karyotype with 2n=11+X originated by centric fusion between 14 autosomal
chromosomes and tandem fusion between one autosomal pair and the long arm of pair 1. A centric
fusion between the X1 and X2 sex chromosomes converted the sex chromosome system into a X type.
Observe the heterozygous state of the centric fusion that originated the chromosomes of pair 1. C.
Karyotype with 2n=10+X, revealing the homozygous condition of the centric fusion of pair 1.
 32

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 37

Os cromossomos de aranhas Theridiidae (Entelegynae): variabilidade

cariotpica interespecfica em Argyrodes e intra-especfica em Nesticodes
rufipes

Leila Miguel Stavale1, Marielle Cristina Schneider1, Antonio Domingos Brescovit2,

Doralice Maria Cella1

1
Departamento de Biologia, UNESP, Rio Claro, So Paulo, Brazil
2
Laboratrio de Artrpodes, Instituto Butantan, So Paulo, So Paulo, Brazil

Palavras-chave: citogentica, meiose, nmero diplide, regio organizadora de

nuclolo, sistema de determinao sexual
 38

Introduo
A famlia Theridiidae uma das mais numerosas dentro da ordem Araneae,
incluindo quase 2.300 espcies descritas taxonomicamente, as quais esto
distribudas em 109 gneros (Platnick, 2009). A grande diversidade de estratgias
de forrageamento e de estilo de vida, que varia de espcies solitrias que no
constroem teias a espcies sociais que exibem cuidado parental, certamente foram
alguns dos fatores que contriburam para o sucesso adaptativo das aranhas
theridiidae (Agnarsson, 2004; Arnedo et al., 2004). Dentro dessa famlia, as espcies
do gnero Argyrodes so conhecidas por seu hbito cleptoparasita, no qual os
indivduos invadem teias de aranhas no relacionadas, geralmente de tamanho
grande, como aquelas do gnero Nephila, em busca de alimento e proteo
(Whitehouse et al., 2002; Agnarsson, 2004). Ainda dentro dessa famlia, a maioria
das espcies do gnero Nesticodes so sinantrpicas, vivendo associadas a
habitaes humanas devido relativa facilidade para obteno de alimento
(Cushingi e Lebeck, 1994).
As famlias Theridiidae e Nesticidae formam o clado das teridioideas, o qual
constitui um ramo derivado dentro de Araneoidea (Griswold et al., 1998).
Citogeneticamente, apenas 29 espcies de Theridiidae pertencentes a 13 gneros
foram analisadas (Arajo, 2007). Com exceo de Argyrodes gazingensis, Chrysso
scintillans e Parasteatoda tepidariorum que exibiram 2n=24 e de quatro
representantes do gnero Latrodectus que apresentaram nmero cromossmico
variando entre 2n=16 a 2n=28, as outras espcies investigadas mostraram o
nmero diplide 2n=22, incluindo um sistema cromossmico sexual do tipo X1X2 e
cromossomos com morfologia acro/telocntrica (Montgomery, 1907; Hackman, 1948;
Suzuki, 1950, 1954; Sokolov, 1960; Diaz e Saez, 1966; Igarashi e Kondo, 1977;
Kageyama e Seto, 1979; Martindale, 1980; Datta e Chatterjee, 1983; Srivastava e
Shukla, 1986; Tugmon et al., 1990; Avils e Maddison, 1991; Gorlov et al., 1995;
Yonju et al., 1995; Chen, 1999; Avils et al., 2000).
Levando-se em conta a enorme diversidade de aranhas Theridiidae e o fato de
nenhuma espcie da fauna Neotropical ter sido examinada sob o ponto de vista
citogentico, o objetivo deste trabalho foi determinar o nmero diplide, o tipo de
sistema cromossmico sexual, a morfologia dos cromossomos, o comportamento
 39

dos cromossomos durante a meiose e o padro de distribuio das regies

organizadoras de nuclolo (RONs) de Argyrodes elevatus Exline & Levi, 1962 e
Nesticopes rufipes (Lucas, 1846). As informaes obtidas foram comparadas com
aquelas de espcies filogeneticamente relacionadas descritas na literatura, visando
estabelecer os mecanismos de evoluo cromossmica que tem ocorrido nesta
famlia.

Material e mtodos
Neste trabalho, uma amostra de 58 indivduos foi analisada, a qual
compreendeu: 13 machos adultos e 13 embries (oito machos e cinco fmeas) de A.
elevatus de Rio Claro (2224S, 4733W), So Paulo (SP), Brasil, 10 embries
(quatro machos e seis fmeas) de A. elevatus de Tup (2156S, 5030W), SP, 12
adultos (cinco machos e sete fmeas) e quatro embries machos de N. rufipes de
Rio Claro, e um macho adulto e cinco embries (dois machos e trs fmeas) de N.
rufipes de Viosa (2045S, 4452W), Minas Gerais (MG), Brasil. Os indivduos
foram identificados taxonomicamente pelo Dr. Antonio Domingos Brescovit, do
Laboratrio de Artrpodes, Instituto Butantan, So Paulo, SP, Brasil.
As preparaes cromossmicas foram obtidas a partir de gnadas de indivduos
adultos e de clulas de embries, utilizando soluo de colchicina 0.16% e 0.05%
respectivamente, de acordo com a metodologia descrita por Arajo et al. (2008). Os
cromossomos foram corados convencionalmente com soluo de Giemsa 3% (47
mL de gua destilada, 1.5 mL de soluo comercial de Giemsa e 1.5 mL de tampo
fosfato pH 6.8) por 15 minutos e posteriormente, impregnados pelo on prata (Howell
e Black, 1980), para a identificao das RONs. A anlise dos cromossomos foi
realizada em microscopia de luz e a imagem das clulas mitticas e meiticas foi
capturada em um fotomicroscpio Olympus BX51, com objetiva 100x de imerso,
optovar 1.6, utilizando o software DP Controller. A morfologia dos cromossomos foi
determinada de acordo com a nomenclatura proposta por Levan et al. (1964).
 40

Resultados
Colorao com Giemsa
Clulas metafsicas mitticas de A. elevatus apresentaram o nmero diplide
2n=21 para os machos e 2n=22 para as fmeas, o sistema cromossmico sexual do
tipo X/XX e a morfologia meta/submetacntrica de todos os cromossomos do
complemento (Figura 1A-B). Os cromossomos autossmicos decresceram
gradualmente em tamanho e o cromossomo sexual X exibiu um tamanho
extremamente grande. Clulas paquitnicas mostraram 10 bivalentes autossmicos
completamente sinapsados e um bloco muito condensado e corado, interpretado
como cromossomo sexual X univalente (Figura 1C). Ncleos em diplteno e
diacinese exibiram at trs bivalentes autossmicos com dois quiasmas terminais,
sendo que os demais bivalentes apresentaram apenas um quiasma intersticial ou
terminal. Nessas fases, o cromossomo X tambm apresentou um alto grau de
condensao e colorao (Figura 1D-E).
A anlise cariotpica de 11 indivduos adultos e 11 embries de N. rufipes
revelou o nmero diplide 2n=22 para os machos e 2n=24 para as fmeas, o qual
condizente com um sistema cromossmico sexual do tipo X1X2/X1X1X2X2 (Figura 2A-
B). Nessa espcie, todos os cromossomos apresentaram morfologia
subtelo/acrocntrica e uma variao gradual em tamanho. Os cromossomos sexuais
foram identificados como elementos de tamanho mediano e com um grau de
condensao um pouco mais elevado que os demais cromossomos do
complemento. Clulas testiculares em paquteno exibiram dois blocos muito
condensados, corados e dispostos lado a lado, confirmando a ocorrncia do sistema
cromossmico sexual do tipo X1X2 para esta espcie (Figura 2C). Nos dipltenos, a
frmula meitica 10II+X1X2 foi observada e todos os bivalentes mostraram apenas
um quiasma intersticial ou terminal. Clulas em metfase II exibiram n=10+X1X2 e
n=10 (Figura 2E).
Um indivduo macho adulto de N. rufipes da populao de Viosa mostrou
nmero cromossmico diplide discrepante daquele encontrado nos outros
exemplares da amostra examinada, ou seja, 2n=24 com cromossomos
subtelo/acrocntricos (Figura 3A). Nessas clulas, os cromossomos sexuais no
apresentaram caractersticas que permitissem diferenci-los dos autossomos.
 41

Ncleos em diplteno e diacinese revelaram a presena de 11 bivalentes

autossmicos e dois cromossomos sexuais X1 e X2 arranjados lado a lado (Figura
3B). Metfases II exibiram dois conjuntos haplides diferentes n=11+X1X2 e n=11
(Figura 3C-D).

Impregnao pelo on prata

Metfases mitticas de A. elevatus submetidas impregnao pelo on prata
revelaram RONs sobre a regio terminal do brao curto dos pares 2, 3 e 4 (Figura
4A-B). Em N. rufipes, as marcaes correspondentes as RONs estavam localizadas
sobre a regio terminal do brao longo do par 4 (Figura 4C-D).
 42

Figura 1 Clulas mitticas (A-B) e meiticas (C-E) de Argyrodes elevatus coradas com Giemsa. A-
B. Caritipos de embries macho (A) e fmea (B), com 2n=20+X e 2n=20+XX, respectivamente.
Observe que o cromossomo X possui tamanho extremamente grande em relao aos autossomos. C.
Paquteno com cromossomo sexual X bem condensado e corado. D. Diplteno, 10II+X, evidenciando
bivalentes com um quiasma intersticial (seta grande) ou terminal (seta pequena). E. Diacinese,
mostrando bivalentes com dois quiasmas terminais (seta). Escala=10m.
 43

Figura 2 Clulas mitticas (A-B) e meiticas (C-E) de Nesticodes rufipes coradas com Giemsa. A-
B. Caritipos de embries macho (A) e fmea (B), com 2n=20+X1X2 e 2n=20+X1X1X2X2,
respectivamente. C. Paquteno, mostrando cromossomos sexuais X1 e X2 heteropicnticos positivos.
D. Diplteno, 10II+X1X2, exibindo bivalentes com um quiasma terminal (seta). E. Metfases II com
n=10+X1X2 e n=10. Escala=10m.
 44

Figura 3 - Clulas testiculares mittica (A) e meiticas (C-D) de Nesticodes rufipes coradas com
Giemsa. A. Metfase com 2n=24. B. Diplteno, 11II+X1X2, exibindo bivalentes com um quiasma
intersticial (seta grande) ou terminal (seta pequena). C-D. Metfases II com n=11+X1X2 e n=11,
respectivamente. Escala=10m.
 45

Figura 4 Clulas mitticas de Argyrodes elevatus (A-B) e Nesticodes rufipes (C-D) coradas com
Giemsa (A e C) e impregnadas pelo on prata (B e D). A. Fmea com 2n=20+XX. B. A mesma clula
apresentada em A, evidenciando RONs sobre a regio terminal do brao curto dos pares 2, 3 e 4
(cabea de seta). C. Fmea com 2n=20+X1X1X2X2. D. A mesma clula mostrada em C, revelando
RONs sobre a regio terminal do brao longo do par 4. Em detalhe, cromossomos do par 4, exibindo
marcaes de RONs bem evidentes. Escala=10m.
 46

Discusso
As caractersticas cromossmicas observadas em A. elevatus foram totalmente
discrepantes daquelas descritas para as 29 espcies da famlia Theridiidae
previamente estudadas, dentre as quais esto cinco outras espcies do gnero
Argyrodes (Arajo, 2007). Dessa forma, o presente trabalho apresenta pela primeira
vez um registro do nmero diplide 2n=21, sistema cromossmico sexual do tipo
X, e todos os cromossomos do complemento com morfologia meta/submetacntrica
para uma aranha Theridiidae. Em contraste, o caritipo 2n=20+X1X2 com
cromossomos subtelo/acrocntricos verificado na maioria dos exemplares da
amostra de N. rufipes, o primeiro representante desse gnero examinado, foi similar
aquele que predominante para a famlia.
Theridiidae pertence ao clado Araneoidea (Griswold et al., 1998), o qual engloba
12 famlias das quais apenas seis foram investigadas citogeneticamente. As aranhas
desse grupo apresentam um caritipo bastante conservado, uma vez que
2n=22+X1X2 com cromossomos acro/telocntricos a formula cariotpica mais
comum em espcies de Araneidae, Linyphiidae, Nephilidae, Tetragnathidae bem
como Nesticidae que grupo-irmo de Theridiidae (Arajo, 2007). Essa ltima
famlia, uma das mais derivadas dentro de Araneoidea, apresenta 80% de suas
espcies com o caritipo 2n=20+X1X2 que diferenciado em relao aos outros
grupos do clado. Essas informaes parecem indicar que a principal tendncia de
evoluo cromossmica, dos grupos basais para os derivados de Araneoidea, foi
atravs de reduo do nmero de autossomos e manuteno do sistema
cromossmico sexual. Em Theridiidae, nmeros diplides mais altos, como 2n=24
verificado em uma espcie de Argyrodes, Chrysso e alguns indivduos de
Parasteatoda radiata, 2n=28, 2n=26, 2n=24 observados em aranhas do gnero
Latrodectus, e nmeros diplides mais baixos, como 2n=16 e 2n=18 tambm
presentes em Latrodectus e 2n=21 encontrado em A. elevatus, provavelmente
tiveram origem a partir do 2n=22.
Em A. elevatus, o sistema cromossmico sexual do tipo X pode ter sido
diferenciado a partir do sistema X1X2 atravs de translocaes Robertsonianas entre
os cromossomos X1 e X2 originalmente acrocntricos. Essa hiptese reforada
pelo fato de que o cromossomo X apresenta dois braos e corresponde ao maior
 47

elemento do caritipo. Alm disso, a converso da morfologia dos autossomos de

acro/telocntrica para meta/submetacntrica provavelmente foi resultado de
inverses pericntricas envolvendo todos os cromossomos do complemento. Por
outro lado, a hiptese de adio de material heterocromtico constitutivo como
mecanismo responsvel por essa converso da morfologia dos autossomos no
pode ser excluda.
Na ordem Arachnida, a reduo do nmero diplide parece ser o principal
mecanismo de evoluo cariotpica (Suzuki, 1954). Tal evento deve ter ocorrido
repetidas vezes durante o processo evolutivo, visto que diferenas inter e intra-
especficas no nmero de cromossomos foram registradas em famlias no-
relacionadas, como Araneidae, Dictynidae, Lycosidae, Oecobiidae, Salticidae,
Sparassidae, entre outras (Arajo, 2007). A converso do sistema X1X2 para o
sistema X tambm parece ser um fato recorrente, pelo menos nas aranhas
Entelegynae, nas quais esse ltimo sistema de determinao sexual o segundo
mais freqente, ocorrendo em cerca de 10% dentre as 590 espcies j estudadas
sob o ponto de vista citogentico (Arajo et al., 2005). Em contraste, a presena de
cromossomos meta/submetacntricos em entelegine bastante espordica e
geralmente tem sido atribuda a rearranjos cromossmicos do tipo fuses cntricas
que envolveram todos os cromossomos do complemento. Essa proposta de
evoluo atravs de fuses do tipo tudo ou nada tem sido corroborada pelo fato de
que espcies que apresentam caritipo saturado por cromossomos de dois braos
tm um nmero diplide mais baixo quando comparadas com outras espcies
intimamente relacionadas e que possuem cromossomos acro/telocntricos (Mittal
1966; Rowell, 1988, 1990, 1991; Amalin et al., 1993; Krl 1995a, b). Dessa forma, o
estudo citogentico de A. elevatus revelou um mecanismo adicional de evoluo
cariotpica para aranhas Entelegynae, o qual possivelmente est relacionado a
inverses pericntricas ou adio de heterocromatina constitutiva em todos os
cromossomos autossomos do complemento.
A grande maioria dos exemplares de N. rufipes estudados exibiu caritipo
semelhante aquele que conservado em Theridiidae. A variao do nmero diplide
observada em um indivduo adulto dessa espcie provavelmente no ocorreu em
virtude de fisses cromossmicas, visto que diferenas marcantes quanto ao
 48

tamanho de um ou mais pares cromossmicos no foram detectadas em todas as

clulas mitticas e meiticas analisadas. Embora pequenas diferenas intra-
especficas no nmero diplide sejam comumente registradas em aranhas (Arajo,
2007), na maioria dos casos, no foram feitas especulaes sobre sua origem. Em
N. rufipes, a variabilidade de nmero diplide certamente est relacionada
presena de um par adicional de cromossomos autossmicos, uma vez que em
clulas em diplteno/diacinese foi invariavelmente verificado 11 bivalentes e em
metfases II conjuntos haplides com n=11+X1X2 e n=11. Considerando que o par
cromossmico extra foi encontrado em todas as clulas do indivduo analisado,
possvel sugerir que sua origem foi a partir de um evento de no-disjuno
cromossmica que ocorreu durante a diviso meitica que formou os gametas de
um de seus parentais. Alternativamente, esse par cromossmico adicional pode
corresponder a cromossomos B que seguem um padro de segregao mendeliana
durante a meiose, caracterstica esta confirmada pelo estudo de clulas em
metfase II. O emprego de tcnicas de identificao de regies cromossmicas
especficas, como o bandamento C e colorao com fluorocromos base-especficos
certamente fornecero subsdios adicionais que podem auxiliar em uma melhor
interpretao dessa variabilidade numrica observada em N. rufipes.
A anlise da distribuio das RONs em espcies de Theridiidae foi realizada
pela primeira vez neste trabalho. A ocorrncia de RONs sobre os autossomos, tal
como verificado em A. elevatus e N. rufipes, parece ser o padro bsico para as
aranhas Entelegynae (Oliveira et al., 2007). Apesar do fato de ambas as espcies
estudadas aqui pertencerem a gneros distintos e exibirem grandes diferenas
cariotpicas, a presena de RONs sobre o par 4 aponta para o fato de que este seja
um padro compartilhado entre espcies desta famlia; no entanto, est proposta s
ser confirmada com a investigao de um maior nmero de espcies. Em A.
elevatus, alm dos rearranjos cromossmicos responsveis pela mudana do
sistema cromossmico sexual e morfologia dos cromossomos, um aumento do
nmero das RONs pode ter ocorrido atravs de eventos de duplicaes seguidos
por translocaes. Uma hiptese similar foi originalmente elaborada por Krl (1995a,
b) para explicar a presena de RONs mltiplas em espcies de aranhas Entelegynae
das superfamlias Amaurobioidea e Dictynoidea.
 49

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Rio Claro, 17 de dezembro de 2009.

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