Table Of Content2006. The Journal of Arachnology 34:435-443
CYTOGENETICS OF THREE BRAZILIAN GONIOSOMA
SPECIES: A NEW RECORD FOR DIPLOID NUMBER IN
LANIATORES (OPILIONES, GONYLEPTIDAE,
GONIOSOMATINAE)
Rosangela Martins Oliveira,^ Adilson Ariza Zacaro,^ P—edro Gnaspini^ and
Doralice Maria Celia': 'Universidade Estadual Paulista UNESP, Institute de
Biociencias, Departamento de Biologia, Caixa Postal 199; CEP —13506-900, Rio
Claro, Sao Paulo State, Brazil; ^Universidade Federal de Vigosa UFV, CCBS,
Departamento de Biologia Geral; Av. PH. Rolfs,—s/n; CEP 36571-000; Vigosa, Minas
Gerais State, Brazil; ^Universidade de Sao Paulo USP, Institute de Biociencias,
Departamento de Zoologia, Caixa Postal 11461; CEP 05422-970, Sao Paulo, Sao
Paulo State, Brazil
ABSTRACT. Currently, 60 species of harvestmen have been karyotyped and all of these are from the
Nearctic and Palearctic regions. This work is the first cytogenetic report of three gonyleptid species of
the suborder Laniatores: Goniosoma aff. badium, G. proximum and G. spelaeum ofthe Neotropicalregion,
fromthe southeastern region ofBrazil. Conventional Giemsastain chromosomepreparations wereobtained
from embryonic cells and adult male testes. Embryo mitotic plates of G. aff. badium and G. proximum
indicated 88 chromosomes, and mitotic spermatogonial plates of G. spelaeum males revealed intra- and
interindividual variation ofchromosomenumber, rangingfrom92-109 chromosomes. Inthethreeanalyzed
species, the mitotic chromosomes were meta- or submetacentric with no obvious sex chromosomes being
identified during mitosis. Prophase I spermatocytes ofG. spelaeum also revealed intra- and interindividual
bivalent numbervariation and furthermore indicated the presence ofmultivalence. The karyotypes ofthese
three Goniosoma species exhibited the largest chromosome pair with a negative heteropycnosis in the
distal region ofthe shortest arm; chromosomes ofG. spelaeum submitted to silverimpregnationevidenced
this negative heteropycnotic region as nucleolus organizer region (NOR). These results, when compared
with cytogenetic data of other Laniatores species from the Palearctic region, indicated that a new record
for diploid chromosome number probably characterize the genus Goniosoma in the Neotropical region.
Keywords: Chiasma, chromosome chain, karyotype, Palpatores, meiosis
Harvestmen are cosmopolitan in distribu- characterized. Overall in the Opiliones, dip-
tion and are grouped into three suborders: Cy- loid numbers vary from 10-78 chromosomes
phophthalmi, with about 50 sparsely distrib- (Sokolow 1929, 1930; Suzuki 1941, 1966,
uted species; Palpatores, with about 2000 1976, 1980; Juberthie 1956; Parthasarathy &
species concentrated in the Holarctic region; Goodnight 1958; Tsurusaki 1982, 1985,
and Laniatores, with about 3500 mainly Neo- 1986a, 1990; Tsurusaki & Cokendolpher
tropical species (Pinto-da-Rocha 1999). 1990; Cokendolpher & Jones 1991). The dip-
Cytogenetic analysis of harvestmen is well loid chromosome numbers found so far in the
documented for the suborder Palpatores with suborder Laniatores ranged from 40 for Pseu-
more than 50 species of Palpatores that have dobiantes japonicus Hirst 1911 (Epedanidae)
been karyotyped. However, the karyotype of to 78 for Vonones sayi (Simon 1879) (Cos-
members of the other suborders are poorly metidae). In contrast, Tsurusaki (1985) found
known with only two species of Laniatores the suborder Palpatores had diploid chromo-
and one of Cyphophthalmi that have been some numbers varying from 10-36. The high-
est diploid chromosome numberof52 was ob-
Corresponding author. E-mail: characid@yahoo. served in the manubriatum (under montanum)
com.br subgroup of the Leiobunum curvipalpe group,
435
436 THE JOURNAL OF ARACHNOLOGY
due to the tetraploidy from specimens having SP All analyzed specimens were collected in
2n = 24 (Tsurusaki 1985). In Cyphophthalmi, November 1999.
Siro nibens Latreille 1804 was found to have To date, G. aff. badium has only been col-
2n = 30 chromosomes (Juberthie 1956). lected from the tunnels ofthe GuaricanaDam;
The moiphology ofthe chromosomes in the G. proximum is widespread in the Atlantic
Opiliones has been described for some Pal- Eorest in the State of Sao Paulo, inhabiting
patores species but for only one Laniatores small cave entrances and trees; G. spelaeum
species. In these specie—s, the predominant is a trogloxene species, living in the cave en-
chromosomes are meta or submetacentric trances in Vale do Ribeira, also in the State of
(Sokolow 1929, 1930; Suzuki 1941, 1966, Sao Paulo (Gnaspini 1995, 1996). These three
1976, 1980; Juberthie 1956; Parthasarathy & species are nocturnal. Male and female vouch-
Goodnight 1958; Tsurusaki 1982, 1985, ers specimens are deposited in the Museu de
1986a, 1990, 1993; Tsurusaki & Cokendol- Zoologia da Universidade de Sao Paulo
pher 1990; Cokendolpher & Jones 1991). (MZSP), Sao Paulo, SP, Brazil.
The sex chromosome system has been mor- The chromosome preparations followed the
phologically distinguished in only 13 species method described by Webb et al. (1978), with
of Palpatores; most of them have an XY/XX some modifications. Dissected embryos and
system (Suzuki 1976, 1980; Tsurusaki 1982, gonads were placed in insect saline containing
1985, 1986a, 1986b, 1989, 1990; Tsurusaki & 0.05% colchicine to arrest the metaphase
Cokendolpher 1990), with one exception, Mi- cells. The hypotonic treatment was completed
topus morio Fabricius 1799, which has a ZZ/ by adding a volume of tap water equal to that
ZW system (Tsurusaki & Cokendolpher of insect saline containing 0.05% colchicine
1990). and was left for 15 min. The material was then
All previous karyotype descriptions of Opi- fixed in methanolic Carnoy for 1 hour. Each
liones were performed in species from Nearctic embryo and/or gonadal tissue was macerated
and Palearctic regions. Moreover, only a few in a drop of 60% acetic acid by tapping the
non-systematic studies were focused on Neo- material with the flat end of metal rod on the
tropical harvestmen (Ramires & Giaretta 1994; slide. The slides were dried on a warm plate
Gnaspini 1995, 1996). This paper describes the (—45° C) for 2 min. The preparations were
first cytogenetic study of harvestmen belonging stained with 3% Giemsa (1.5 ml of Giemsa
to suborder Laniatores (Gonyleptidae, Gonio- stock solution, 45 ml of distilled water, and
somatinae) from the Neotropical region; the 1.5 ml ofpH 6.8 phosphate buffer). Some tes-
study includes embryonic metaphase analyses ticular preparations of G. spelaeum were sub-
of Goniosoma aff. bcidium and G. proximuin mitted to silver impregnation (Howell &
(Mello-Leitao 1933) and analyses of testicular Black 1980; Kodama et al. 1980) to determine
mitotic and meiotic cells ofG. spelaeum (Mello- the ehromosomes bearing the nucleolus or-
Leitao 1933). The goal of this work was to de- ganizer regions (NORs).
termine the karyotypes of these three Gonioso- The egg samples of G. aff badium and G.
ma species and additionally, to describe the proximum indicated asynchronous embryo de-
chromosome behavior of G. spelaeum during velopment and only a few embryos were suit-
meiosis. able for cytogenetic analysis. The first cyto-
genetic results of G. spelaeum revealed
METHODS
variable diploid chromosome numbers, which
Cytogenetic analyses were carried out on was initially interpreted as a consequence of
three Goniosoma species from southeastern technical preparation problems. Considering
Brazil: on six embryos of G. aff. badium from that the loss and overlapping ofchromosomes
Barragem de Guaricana (25°43'S, 48°58'W), could be a result of, respectively, long and
Sao Jose dos Pinhais, PR; on eight embryos short duration of hypotonic treatments, de-
of G. proximum from Gruta do Moquem, PE- creasing times of hypotonic treatment were
TAR-Parque Estadual Turistico do Alto Ri- tried and also resulted in diploid number var-
beira (24°38'46"S, 48°42'2"W), Iporanga, SP; iation. Finally, the squash technique was also
on six adult specimens (four males and two tried to avoid putative overspreading and loss
females) of G. spelaeum from Gruta do Tatu of chromosomes of the mitotic and meiotic
(24°16 05"S, 48°25'()3"W), Ribeirao Grande, plates. Due to the presence of high diploid
OLIVEIRA ET AL.—CYTOGENETICS OF GONIOSOMA 437
it It mitotic plates showing low numbers of over-
lapped chromosomes and those having a rea-
sonable degree of chromosome condensation
to allow centromere identification.
RESULTS
The majority ofembryo metaphases ofG. aff.
56789
II t2il3l4If if Ilia II li II badium and G. proximum revealed a karyotype
I 10 of 2n = 88 chromosomes (Figs. 1, 2); in a few
IIII I12I I13I 1141 a^5«in«” 1171 I18I I\9I I20I embryo metaphase cells, the diploid chromo-
11 II II II II •• some number varied in the same individual or
different specimens, which was interpreted as
31 32 34 35 36 37 38 39 accidental gaines or losses of chromosomes
41 42 44 2 among cells during slide preparation. From a
total of 76 spermatogonial metaphases prepara-
1 2 5 6 7 tions of G. spelaeum, only 26 were suitable for
cytogenetic analysis. The selected G. spelaeum.
11 13 16 17 18 19^ 20 spermatogonial metaphases showed intra- and
21 22 23 24 25 26 •27t m28m «2•9 i30t interindividual variation in relation to the dip-
31 32 33 34 35 36 37 38 39 40 loid chromosome number that ranged from 92-
41 42 43 44 45 46 47 4S 49 50^ 109 chromosomes (Table 1, Fig. 3). Considering
%9 it m the high chromosome number variation, the dip-
52 53— loid number for the G. spelaeum was not deter-
Figures 1-3. Karyotypes of three Goniosoma mined. The chromosomes of these three Gon-
species stained with Giemsa; 1. Goniosoma aff. iosoma species exhibited mainly meta- or
mbadauudlmitumemamlbeermyb(2orny(o=2n(120=n5).8=8A)r;8r8)o3;.wsG2.oinnGdiioocnasitoeomstaohemsanpeegplaratoeixuivm-e msuobsmoemtaecsentthraitcwemroerpdhifofleorgenyti(aFtiegds.by1-s3i)z.e aCnhdr/oo-r
heteropycnotic region ofpair 1. Scale bar = 10 pm. cmaotrephsoelxogcihcraolmhoestoemroegse,newiteyr,ewnhoitchnoctoeudldinintdhie-
karyotypes of the Goniosoma species.
chromosome number and the small size ofthe A notable karyotypic feature shared by
chromosomes, the squash technique did not these Goniosoma species was the presence of
allow any better resolution of the mitotic a negative heteropycnosis in the distal region
plates and also indicated chromosome number of the short arm of the largest chromosome
variation. Female gonads ofG. spelaeum were pair of the karyotype (Figs. 1-4). Other dif-
also analyzed for chromosome preparations, ferentially stained chromosome regions were
but no cellular division was found. also noted in these karyotypes. However,
Due to the high number and small size of these regions did not constitute common char-
the chromosomes, some restrictions were es- acteristics to the three analyzed Goniosoma
tablished to accomplish the karyotype char- species. The identification of all these differ-
acterization ofthese three Goniosoma species. entially stained regions depended on the de-
This included considering only well spread gree of chromosome condensation.
—
Table 1. Number of chromosomes observed in diploid cells (2n) and first meiotic metaphases (n) of
four males of Goniosoma spelaeum.
2n n
Male
number # of preps Range mean mode median # of preps range mean mode median
1 6 92-103 97.5 96 97.5 6 39-46 42 40 40.5
2 6 93-105 96.8 97 96 7 40-47 43.6 42 42
3 5 97-100 98.6 98 98 1 43-43 43 43 43
4 9 99-101 103.1 101 101 8 44-53 47 45 45.5
^
—
438 THE JOURNAL OF ARACHNOLOGY
T’
Silver impregnation of Giemsa stained mi-
V totic metaphases of G. spelaeum evidenced
NOR
active in the distal region of the short
It It arm of the pair 1 chromosomes (see arrow in
Figs. 5, 6). The NOR labeling was coincident
with the negative heteropycnotic region of
1 1 1 pair 1.
Testicular preparations of G. spelaeum in-
4
dicated cells in pachytene and diplotene stag-
es. Pachytene cells of G. spelaeum showed at
least 46 filamentous structures, regular in
width, which were probably formed by sepa-
rated or end-to-end associated bivalents (Fig.
7); nevertheless, the exact number of associ-
ated chromosomes was impossible to deter-
mine.
Diplotene spermatocytes of G. spelaeum
also revealed variable bivalent numbers and
additionally showed the occurrence of a typ-
ical chromosome chain. The diplotene cells
exhibited a maximum of 53 bivalents plus a
chain (Figs. 8, 9). From 100 studied diplotene
cells, only 24 permitted a count ofthe bivalent
number and clearly showed the chromosome
chain. Table 1 shows the bivalent number var-
iation in the analyzed diplotene cells. Early
diplotene cells exhibited ring multivalence
(Fig. 8 and late ones showed a linear multi-
)
valence (Fig. 9). Probably the latest multiva-
lent configuration appeared due to the preco-
cious chiasma terminalization (Fig. 9).
Although sufficient number of diplotene
cells have been analyzed, the number ofchro-
mosomes in the multivalent was not deter-
mined because the chromosomes were highly
condensed, and lacked morphological resolu-
tion. Additionally, the precocious chiasma ter-
minalization events, involving the chromo-
somes in the multivalent and perhaps the
H— bivalents, allied with the high degree of chro-
mosome condensation, also compromised the
6 establishment of the exact bivalent number.
— As a consequence, some diplotene chromo-
Figures 4-6. Mitotic chromosomes of Gonio-
some elements could not be identified as uni-
soma spelaeum: 4. Pairs 1 submitted to Giemsa
staining showing different degrees of condensation valent or bivalent. Moreover, the type sexual
and clearly visible negative heteropycnotic regions determination system was not established,
(arrows); 5. Spermatogonial metaphase (2n = 100) considering that none of the meiotic chromo-
stained with Giemsa; 6. The same spermatogonial some elements showed any particular feature
metaphase showed in Fig, 5 submitted to silver ni- that could permit sex chromosome recogni-
trate impregnation, exhibiting NORs (arrows) in the tion. Giemsa stained interphasic nuclei of G.
pair 1 chromosomes. Scale bar = 10 fxm. spelaeum indicated several positive hetero-
pycnotic dots, which were variable in number
and probably representative of heterochroma-
tin. Therefore, neither large nor frequent het-
OLIVEIRA ET AL.—CYTOGENETICS OF GONIOSOMA 439
eropycnotic blocks were noted in interphasic
nuclei that could be interpreted as sex chro-
matin.
Early prophase I meiocytes and interphasic
nuclei of G. spelaeum submitted to the silver
impregnation revealed the presence of one to
three blocks of nucleolar material deeply im-
pregnated (Figs. 10-13), suggesting that there
are at least one and a maximum ofthree active
NORs. Late diplotene cells did not provide
detectable labeling ofnucleolar material or bi-
valent carrier of the NORs.
DISCUSSION
The karyological results ofthese three gon-
yleptid species are similar to Vonones sayi
(Simon 1879) (Laniatores, Cosmetidae) in re-
lation to the high number and morphology of
the chromosomes and no obvious sex chro-
mosomes. V. sayi has 2n = 78, with most of
the chromosomes being metacentric or sub-
&
metacentric (Cokendolpher Jones 1991).
However, the high chromosome numbers ob-
served in these species that belong to the sub-
order Laniatores, contrast with those de-
scribed for most of the species of Palpatores,
whose diploid chromosome numbers vary
from 10-36 (Tsurusaki 1986a; Tsurusaki &
Cokendolpher 1990).
In comparison to the total number of spe-
cies of Opiliones, only a small percentage
have been karyotyped, and those are members
of Laniatores. In considering the cytogenetic
data described for Cyphophthalmi, Palpatores,
and Laniatores species, the Goniosoma spe-
cies karyotype differentiation seems to have
occurred by an increase of the chromosome
number. Probably chromosomal centric fis-
sions followed by pericentric inversions were
responsible for the chromosome number in-
crease and meta- or submetacentric morphol-
ogy maintenance of the Goniosoma species.
Using phylogenetic analyses based on mor-
<—
rows indicate linear chromosome association; 8.
Diplotene cell with approximately 45 bivalents plus
a ring multivalent (large arrow). The small arrows
indicate abivalent with interstitial and terminalchi-
— asmata; 9. Late diplotene cell with about 47 biva-
Figures 7-9. Goniosoma spelaeum prophase I lents plus alinearmultivalent (arrowhead). Theme-
cells stained with Giemsa: 7. Pachytene cell with dium size arrow indicates a bivalent with one
approximately 45 filamentous structures. The ar- interstitial chiasma. Scale bar = 10 gm.
440 THE JOURNAL OF ARACHNOLOGY
—
Figures 10-13. Prophase I and interphasic nuclei of Goniosoma spelaeum submitted to silver nitrate
impregnation: 10,11. Early prophase I and diplotene nuclei, respectively, both exhibiting three blocks of
nucleolar material (nu); 12,13. Interphasic nuclei, showing respectively, two and three blocks ofnucleolar
material (nu). Scale bar = 10 p.m.
phological and molecular data from several somes for translocation or supernumerary
species belonging to Cyphophthalmi, Palpa- chromosomes, or random gaines or losses of
tores, and Laniatores, Giribet et al. (1999) chromosomes among cells during the proce-
found that Gonyleptoidea (Laniatores) in- dures of chromosomal preparations.
cludes species with highly derived character- The variation in intra-individual chromo-
istics. This differs from other species of the some number is not common, but there are
Laniatores superfamily. These data corrobo- descriptions about its occurrence in some spe-
rate the possibility of the Goniosoma species cies that possess heterozygous specimens for
having a highly derived karyotype. structural chromosome rearrangements (Ohno
The diploid number variation noted in the et al. 1965; Begak et al. 1966; Hartley &
G. spelaeum testes cells could be a conse- Horne 1984; Thode et al. 1985; Giles et al.
quence of differential chromosome non-dis- 1985). This intra-individual variation seems to
junction, involving heterozygous chromo- be a consequence of somatic chromosome
OLIVEIRA ET AL.—CYTOGENETICS OF GONIOSOMA 441
non-disjunction that could favor the reconsti- ated with a single A-chromosome. Although
tution of homozygous chromosomes. The this species has an XY-XX sex chromosome
chromosome non-disjunction presumably system (Tsurusaki 1993), whether this single
functions as an “accumulation mechanism” NOR is on one of the sex chromosomes in
and is particularly evident in some polymor- unclear. In G. spelaeum, the NORs were as-
phic species for supernumerary chromosomes sociated with the largest pair, which could
(White 1973; Gorlov & Tsurusaki 2000a, b; possibly be sex chromosomes. In Palpatores
Tsurusaki & Shimada 2004). harvestmen species, whose sex determination
The presence of supernumerary chromo- system has already been established (Tsuru-
somes, which are extra chromosomes, in Op- saki 1985, 1989, 1990, 1993; Tsurusaki &
iiiones is well documented in Psathyropus Cokendolpher 1990) the X and Z sex chro-
tenuipes Koch 1878 (formerly Metagagrelia mosomes displayed similar size to the largest
tenuipes Suzuki 1949) (Tsurusaki 1993; Gor- chromosomes of the diploid complement.
& &
lov Tsurusaki 2000a, b; Tsurusaki Shi- Considering the range ofnucleolar material
mada 2004). This species showed extensive observed at ioterphase and prophase I, it is
variation in the number of chromosomes, likely that more than two NOR-bearing chro-
ranging from 0-19, am.ong cells of a single mosomes might be present, since activation or
individual and among individuals of a single inactivation mechanisms in these regions
population, as well as among populations. could be playing a role on the gene transcrip-
They behave as univalents during meiosis, tion.
though some of them seem to form a chain This work characterized three species of
v/ith end-to-end associations in diakinesis Brazilian harvestmen G. aff. badium (2n =
(Gorlov & Tsurusaki 2000b). 88), G. proximum (2n = 88) and G. spelaeum
The G. spelaeum multivalence was identi- (2n = 92“109). These species showed a high
fied in all prophase I cells analyzed, but this chromosome diploid number and several
feature does not constitute an accurate param- meta- or submetacentric chromosomes. The
eter to determine ifthese chromosomes are or results suggests that chromosomal evolution
are not supernumeraries. The description of in the genus Goniosoma occurred by an in-
multivalent-like associations between super- crease of chromosomal number through cen-
numerary chromosomes during meiosis are tric fissions, which were followed by pericen-
&
rare in the literature (Jones Rees 1982; Gor- tric inversions. G. spelaeum evidenced both
lov & Tsurusaki 2000b). intra- and interindividual variable diploid
The G. spelaeum meiotic multivalent con- numbers which could be explained by the
figurations suggest the occurrence of hetero- presence of translocated chromosomes in het-
zygous chromosomes for serial translocations. erozygous. The presence of multivaleets dur-
Although pachytene filaments with special ing meiosis in this species corroborates the
configurations, such as loops or open crosses, hypothesis of occurrence of heterozygous
are indicative of heterozygosity for structural chromosomes for serial translocations.
chromosome rearrangements, these were not ACKNOWLEDGMENTS
seen in the analyzed cells, probably due to the
occurrence of heterosynapsis. The G. spe- The authors thank Dr. Sanae Kasahara and
laeum multivalent configuration is similar to Dr. Ana Paula Zampieri Silva, from the De-
that found in Delena cancerides Walckenaer partameeto de Biologia, IB, UNESP, Rio Gla-
1837 (Araneae), Neotermes fulvescens (Sil- re, Sao Paulo State, Brazil, forthe suggestions
vestri) 1901 (Isoptera) and Keyacris scurra given during the developm,ent of this work
(Rehn) (Orthoptera), which arose from hetero- and fortheircritical reading ofthe manuscript.
This work was supported by FAPESP (Fun-
zygous and serial interchanges, such as centric
dagao de Amparo aPesquisado Estado de Sao
fusions and reciprocal translocations (Rowell
1985, 1991a, 1991b; Hancock & Rowell Paulo, process number01/12464-9) and CNPq
1995; Martins & Mesa 1995; White 1973). (Conselho Nacional de Desenvolvimento
In Opiliones there is only one description Cientifico e Tecnologico).
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