Table Of Content2008. The Journal ofArachnology 36:453-456
Mesaholivar hvasiliensis (Moenkhaus 1898) and Mesaholivar cyaneotaeniatiis (Keyserling 1891)
(Araneomorphae, Pholcidae): dose relationship reinforced by cytogenetic analyses
Manuela Oliveira Ramalho', Douglas Araujo--^, Marielle Cristina Schneider', Antonio Domingos BrescoviC and Doralice
Maria Celia': 'Universidade Estadual Paiilista, Instituto de Biociencias, Departamento de Biologia, UNESP, Avenida
24=A, 1515, Bela Vista, CEP. 13506-900, Rio Claro, Sao Paulo, Brazil; ^Universidade Estadual de Mato Grosso do Sul,
UEMS, Unidade Universitaria de Mundo Novo, BR 163, Km 20.2, Universitario, CEP. 79980-000, Mundo Novo,
Mato Grosso do Sul, Brazil; -''Instituto Butantan, Laboratorio de Artropodes, Avenida Vital Brasil, 1500, CEP. 05503-
900, Sao Paulo, Sao Paulo, Brazil
Abstract. Pholcidae is the most diverse family among haplogyne spiders but only 15 species have been analyzed
cytogenetically. Thesestudiesrevealed that thediploid numbervariesfrom 2n = 15 to 2n = 32, that therearethreetypesof
sex chromosome systems in males (X, XiXt and X1X2Y), and that the chromosomes are predominantly biarmed. Within
the genus Mesaholivar, only Mesaholivar hiteus (Keyserling 1891) has been karyotyped, and it showed 2n = 15 = 14 + X,
with all chromosomes being metacentric. In the present work, we characterize the mitotic and meiotic chromosomes of
Mesaholivarhrasiliensis(Moenkhaus 1898)and Mesaholivarnw;comen (Keyserling 1891). Malemitoticmetaphasesof
thetwo speciesshowed thediploid number2n = 17 = 16+X; oogonialcells ofM. hrasiliensisshowed 2n = 18 = 16+XX.
In both species, the chromosomes were exclusively biarmed, and the X chromosome was the largest element of the
karyotype. Diplotene spermatocytes of the two species exhibited 8II X and the occurrence of only one terminal or
interstitialchiasma perbivalent. In M. cyaneotaeniaus, metaphases II with n = 9 = 8 +X and n = 8 werefound, indicating
theregularsegregation ofallchromosomesduringmeiosis I. MitoticmetaphasesofM. hrasiliensisstainedwith CMA3/DA/
DAPI revealedGC-richchromatin in theterminal region ofalmost allautosomes,especiallyin pair2. Anearlierrevision of
the New World pholcids grouped M. hrasiliensis and M. cyaneotaeniatiis in a “southern group” and placed M. hiteiis in a
“miscellaneous group.” A molecular study showed a closer relationship between M. hrasiliensis and M. cyaneotaeniatiis
than between M. hiteus and either of these two species. The 2n = 17 found in M. hrasiliensis and M. cyaneotaeniatiis
corroborates this hypothesis, given that M. hiteus has a diploid number of2n = 15.
Keywords: Chromosome, Haplogynae, meiosis, sex chromosome system, spider
According to Platnick (2008), the family Pholcidae Koch chromosomally and its diploid complement was described as
1851 includes 999 extant species, and thus constitutes the most 2nJ = 15 = 14 + X (Araujo et al. 2005). Considering that M.
diverse family among haplogyne spiders. A phylogenetic hrasiliensis and M. cyaneotaeniatiis belong to the same
analysis based on morphological characters separated pholcids Mesaholivar operational group, the aim of the present work
into four clades: “ninetines,” “pholcines,” “holocnemines,” was to determine and compare the mitotic and meiotic
and the “New World clade” (Huber 2000). The genus chromosomal characteristics of both species and to discuss
Mesaholivar Gonzalez-Sponga 1998, which ranges from the cytogenetic similarities with M. hiteus, the karyotype of
northern South America to northern Argentina, is included which was previously described by Araujo et al. (2005).
in the “New World clade” and possesses 45 species (Huber METHODS
2000, 2008). Moreover, the genus is divided into four
operational groups based on morphological characters: a Three embryos (one male and two females) and six adults
“northern group with spines on male metatarsi,” a “northern (five males and one female) of M. hrasiliensis collected at
group without spines on male metatarsi,” a “southern group,” Estagao Ecologica de Boraceia (22°11'S, 48°46'W), Sales-
and a “miscellaneous group.” The species Mesaholivar opolis, state of Sao Paulo, Brazil and five males (two pre-
hrasiliensis (Moenkhaus 1898) and Mesaholivar cyaneotaenia- adults and three adults) of M. cyaneotaeniatiis collected at
tus (Keyserling 1891), both part ofthe “southern group,” are Universidade Estadual Paulista (UNESP), Rio Claro
distributed in southern and eastern Brazil and northern (22°41'S, 47°56'W), state of Sao Paulo, Brazil, were cytoge-
Argentina (Huber 2000, 2008). netically analyzed. The pre-adult and adult specimens were
Until now, only 15 pholcid species have been analyzed deposited in the collection ofArachnida in the Laboratorio de
cytogenetically, and their diploid number varies from 2n3' = Artrdpodes, Instituto Butantan, Sao Paulo, state of Sao
15 to 2n^ = 32. The sex chromosome system (SCS) is Paulo, Brazil (IBSP, A.D. Brescovit) (M. hrasiliensis - IBSP
commonly of the X type, but SCS of the X1X2 and XiX^Y 48221, 48223, 48229, 48234, 48235, 48241; M. cvaneotaeniatus
types were also recorded. The meta/submetacentric morphol- - IBSP 75519, 75524, 7551 1, 75512, 75513).
ogy of the chromosomes predominates in the species Embryos and gonads were dissected in insect physiological
karyotyped (see Oliveira et al. 2007). Mesaholivar hiteus solution (7.5g NaCl, 2.38g Na2HP04, 2.72g KH2PO4 in IL of
(Keyserling 1891), a representative of the “miscellaneous distilled water), transferred to colchicine solution (0.05% for
group,” is the single species of Mesaholivar characterized embryos and 0.16% for gonads, both in insect physiological
solution) and left for 2 h; a volume ofhypotonic solution (tap
“Corresponding author. E-mail: daraujo(^uems.br water) equal to that ofthe colchicine solution was added, and
453
454 THE JOURNAL OF ARACHNOLOGY
#
If fl II ft II tf If It 1
a 1 2 3 4 5 6 7 8 X
If li II It II it II li II
b 1 2 3 4 5 6 7 8 X
X
h U
K >
If
II ..
c “I 2 3 4 5 6 7 8 X
Figure 1.—Karyotypes ofthe Mesabolivar species standard stained with Giemsa: a, b. Mesaholivar hnisiliensis with InS = 17 = 16 + X and
2n9 = 18 = 16+XX, respectively;c. Mesaholivarcyaneotaeniatuswith2nJ = 17 = 16+X. Notethat in bothspeciestheXsexchromosomeisthe
largest element ofthe complement. Scale = 10pm.
after 15 min, the material was placed in Carnoy I (3 methanol: taeniatus, the male mitotic metaphases presented a less
1 acetic acid) fixative solution for 30 min. The material was condensed condition, becoming impossible to determine the
then transferred to a drop of60% (embryos) or 45% (gonads) precise position of the centromere in the majority of the
acetic acid on a microscope slide, and the material was chromosomes (Fig. Ic). However, the analysis ofmetaphaseII
macerated to form a cell suspension. The slide was dried on a cells of M. cyaneotaeniatus revealed the meta/submetacentric
metal heating plate at 35^0° C and most ofthem were stained morphology of all chromosomal elements (Fig. 2c, d). No
with a 3% Giemsa solution for 13-15 min. Additionally, for evident secondary constrictions were found in mitotic meta-
detection of the AT- and GC- rich chromatin regions, some phase chromosomes ofeither species.
chromosomal preparations were stained with the lluoro- The observation ofdiplotene cells ofM. brasiliensis and M.
chromes 4'-6-diamidino-2-phenylindole (DAPI) and chromo- cyaneotaeniatus revealed eight autosomal bivalents and one
mycin A3 (CMA3), and counterstained with distamycin A univalent, the X chromosome (8II-1-X), identified by its large
(DA), according to the technique described by Schweizer size. In both species, each autosomal bivalent presented only
(1980). The cells stained with Giemsa were photographed one terminal or interstitial chiasma (Fig. 2a, b). The charac-
under a Zeiss microscope, using a Kodak Imagelink HQ teristics shown by diplotene cells of the two Mesabolivar
Microfilm, and those stained with DAPI and CMA3 species studied here confirmed thediploid numberof2n3' = 17
fliiorochromes were photographed under a Leica fluorescence and the SCS of the X/XX type in males and females,
microscope, using a Kodak T-Max 100 Film. The chromo- respectively. The metaphase II cells of M. cyaneotaeniatus
somal morphology was determined following the nomencla- exhibited nine and eight chromosomes (Fig. 2c, d). The cells
ture proposed by Levan et al. (1964). with nine chromosomal elements included the X chromosome
(n = 9 = 8 + X) that was always easily recognized by its large
RESULTS
M size and positive heteropycnosis (Fig. 2c); the cells with eight
The analysis of mitotic metaphases of male brasiliensis elements (n = 8) possessed only autosomes (Fig. 2d). These
and M. cyaneotaeniatus revealed the karyotype with 2n = 17 characteristics indicated the regular and reductional segrega-
= 16-I- X (Fig. la, c). Only female specimens ofM. brasiliensis tion of all chromosomes during anaphase 1.
were available for the study and showed a diploid complement Due to the limited material available, only mitotic
of 2n = 18 = 16 + XX (Fig. lb). There were no evident metaphases of M. brasiliensis were stained with base-specific
chromosomal sizeclasses. Based on chromosomemorphology, fluorochromes. Mitotic chromosomes subjected to DAPI
the elements of the karyotype were tentatively arranged in appeared to stain homogenously, with no differential bright
pairs in order of decreasing size. In both species, the X region. On the other hand, the same chromosomes stained
chromosomewasalwayseasily identified as thelargest element with CMA3 showed bright fluorescence in the terminal
ofthe karyotype (Fig. 1). In M. brasiliensis, the chromosomal region in the majority of the chromosomes, especially in
morphology was established by means of embryo mitotic the elements of pair 2. The X chromosomes were one
metaphase chromosomes, and all elements were classified as of the exceptions, without differentially fluorescent regions
meta/submetacentric (Fig. la, b). In the case of M. cyaneo- (Fig. 3).
RAMALHO ET AL.—CYTOGENETICS OF TWO MESABOLIVAR SPECIES 455
a b
V
c d
—
Figure 2. Testicular meiocytes of the MesahoUvar species standard stained with Giemsa: a, b. Diplotenes of Mesaholivar hrasiHensis and
MesabolivarcyaneotaenMiatus, respectively, bothwith 8II+X,exhibitingoneinterstitial (arrow)oroneterminal(arrowhead)chiasmaperbivalent;
c,d. Metaphases II of cyaneotaeniatuswith n = 9 = 8+Xand n = 8chromosomes, respectively, revealingthatallchromosomesarebiarmed.
Scale = 10pm.
DISCUSSION Oliveira et al. 2007). Concerning thediploid number, 2n^ = 17
was first described for the pholcid Micropholcus fauroti
TheMgeneral chromosomal characteristics of M. hnisiliensis (Simon 1887) (Araujo et al. 2005). However, the present
and cyaneotaeniatus agree with those found in other addition of two more pholcids with the same number of
pholcid spiders that have been analyzed cytogenetically (see chromosomes indicates that this diploid number could be as
Figure 3.—Mitotic metaphase ofMesaholivarbrasiliensis with 2n? = 18 = 16+ XX stained with CMA3 GC-specific Oiiorochrome, showing
brightfluorescenceintheterminal regionofthemajorityoftheautosomes.ThearrowsindicateprominentGC-rich DNA sequencesin thepair2
chromosomal elements. Scale = 10pm.
456 THE JOURNAL OF ARACHNOLOGY
widespread as the 2n^ = 15, which has been recorded from M. luteus. The revision by Huber (2000) indicated a probable
five species of the family. close relationship between Mesaholivar luteusand Mesaholivar
With regard to the SCS, the X type in males and XX in levii Huber 2000, both belonging to the “miscellaneous
females found in both species here studied was also group.”
encountered in nine other pholcids (Bole-Gowda 1958; ACKNOWLEDGMENTS
Cokendolpher 1989; Xiuzhen et al. 1997; Araujo et al. 2005;
Krai et al. 2006; Oliveira et al. 2007), and thus may be the This research was supported by Fundaqao de Amparo a
most prevalent form in this family. In species of Pholcidae Pesquisa do Estado de Sao Paulo (FAPESP - 06/53275-3),
with X/XX system, the X chromosome seems to be the largest Coordenaqao de Aperfeiqoamento de Pessoal de Nivel
or nearly the largest element of the karyotype. This fact may Superior (CAPES), and Conselho Nacional de Desenvolvi-
indicate a single origin ofthis SCS in this group ofspiders. An mento Cientifico eTecnologico (CNPq-Grant 301776/2004-0
elegant hypothesis for the origin of the X system from the to ADB).
X1X2Y SCS in pholcids was already elaborated by Krai et al. LITERATURE CITED
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