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1290 Mantovani
exchange and sequence homogeneity, the former acting
as a long-range homogenization force. Obviously, rates
of exchange cannot be too low relative to mutation rate
to maintain the commonly observed high degree of in-
traspecific sequence homogeneity (see Stephan 1997).
On the other hand, concerted evolution of ribosomal
DNA arrays in parthenogenetic lizards of the Heteron-
otia binoei complex is consistent with biased gene con-
version as the operative mechanism (Hillis et al. 1991).
SatDNA studies have been successfully used as a
tool for taxonomic and phylogenetic investigations with-
in many invertebrate groups (Sainz and Cornudella
1990; Badaracco et al. 1991; Bachmann and Sperlich
1993; Rovira, Beerman, and Edstrom 1993; La Volpe
1994; Bachmann, Venanzetti, and Sbordoni 1996; Ugar-
kovic, Podnar, and Plhol 1996). The molecular analyses
of Bacillus genomes showed the existence of a Bag320
satDNA family in B. atticus and B. grandii and its ab-
sence in B. rossius (Mantovani et al. 1993, 1997). Nu-
cleotide sequences from B. atticus and B. grandii cluster
conspecifically in phylogenetic dendrograms. Moreover,
while the nucleotide sequences of the three B. grandii
subspecies build up subspecific clusters, B. atticus
clones intermingle with no geographical trend. The ab-
sence of intrasubspecific higher sequence similarity of
the Bag320 satDNA in B. atticus has been related to its
parthenogenetic reproduction and its automictic game-
togenesis (Mantovani et al. 1997).
The present paper reports a detailed study of the
specific Bag320 family in the unisexual B. lynceorum to
verify the contribution of both B. atticus and B. grandii
to its hybrid origin and, if it can be verified, to analyze was chosen as the basic estimate of the proportion of
sequence variation in an apomictic system.
Materials and Methods
Sampling
Individuals belonging to distinct populations of B.
lynceorum stick insects were collected during 1990–
1994 from the following Sicilian locations (fig. 1): Cas-
sibile (1; Cas), Catania (2; Cat), Mascalucia (3; Mas),
Melilli (4; Mel), Noto (5; Not), Ponte Diddino (6; Pdd),
Ragusa (7; Rag), Siracusa (8; Sir), Vendicari (9; Ven),
Villasmundo (10; Vil), and Vittoria (11; Vit).
Field-collected specimens were reared in the labo-
ratory in aerated cages on fresh food plants (bramble or
lentisk). Bodies were frozen and stored at Ϫ80ЊC until
they were used for molecular investigations.
Cloning of Satellite DNA
Genomic DNA was prepared from single animals,
according to the method described by Preiss, Hartley,
and Artavanis-Tsakonas (1988). Genomic DNA of B.
lynceorum from Cassibile was screened for restriction
analysis by endonuclease digestion and subsequent gel
electrophoresis. A ladderlike pattern of prominent bands
indicated the presence of a tandemly repeated satDNA.
The following restriction enzymes were applied: AluI,
BclI, BglII, ClaI, EcoRI, HaeIII, MspI, NdeI, PstI, SalI,
SmaI, and TaqI. The genomic DNAs of all other B. lyn-
ceorum populations were restricted only with BglII.
BglII fragments were extracted from agarose gels using Downloaded from http://mbe.oxfordjournals.org/ by guest on April 9, 2016
the Jetsorb kit (Genomed), ligated to the pUC19 plasmid
vector (BRL), and used to transform E. coli DH5␣ com-
petent cells (Gibco). Clones were screened using the ␤-
galactosidase gene blue-white color system (Sambrook,
Fritsch, and Maniatis 1989), and recombinant ones were
identified through Southern blot hybridizations using a
mixture of pAAT/Ven1 and pGG/Not2 plasmids (Man-
tovani et al. 1997) as probe. In particular, DNA was
transferred to nylon membranes (Boehringer Mannheim)
according to the protocol of Southern (1975). Labeling
of probe DNA, filter hybridizations, and detection of the
hybridization signals were performed as described in the
manual for the nonradioactive DIG DNA labeling and
detection kit (Boehringer Mannheim). PAAT/Ven1 and
pGG/Not2 plasmid DNA was obtained using the Flex-
iprep Kit (Pharmacia). Both strands of at least two
clones from each population were sequenced by the di-
deoxy-chain termination method (Sanger, Nicklen, and
Coulson 1977) using the Autoread sequencing kit and
the A.L.F. automatic sequencer (Pharmacia).
DNA Sequence Analysis
Nucleotide sequences were aligned using the clus-
tal algorithm of the Sequence Navigator program (ver-
sion 1.0.1, Applied Biosystems Inc.). Genetic distances
were calculated between all pairs of clones according to
Kimura’s (1980) two-parameter method. Dendrograms
were constructed from the matrix of genetic distances
using the neighbor-joining algorithm in the software
package MEGA (version 1.0) (Kumar, Tamura, and Nei
1993). The p-distance (Kumar, Tamura, and Nei 1993)
different nucleotide sites between each two sequences
and was analyzed with a Student’s t-test for independent
samples. For appropriate comparisons, the following
clones of B. atticus and B. grandii were taken into ac-
count: pAAT/Ven1 (B. atticus atticus, Vendicari);
pAAT/Epi2–4 (B. atticus atticus, Epidavro); pACY/
Epk1, 4, 12 (B. atticus cyprius, Episkopi); pACA/Igo2–
4 (B. atticus carius, Igoumenitsa); pGG/Not2, 4, 10, 17,
20 (B. grandii grandii, Noto); pGB/Sco1, 2, 4, 7, 8, 10
(B. grandii benazzii, Scopello); pGB/Lev1, 4, 5 (B.
grandii benazzii, Levanzo Island); pGM/Mar9, 10, 12
(B. grandii maretimi, Marettimo Island) (Mantovani et
al. 1997; accession numbers U92526 and U79495–
U79520).
Results
Genomic DNA restriction analysis in the Cassibile
population showed a typical ladderlike pattern, with
some stain intensity differences, by AluI, BclI, BglII,
ClaI, MspI, and TaqI enzymes. Filter hybridization ex-
periments identified the ladders as pertaining to the
Bag320 satellite family.
According to the restriction data, the approximately
300-bp fragments obtained in BglII-restricted DNAs of
all B. lynceorum populations were regarded as mono-
mers and cloned. Thirty-four clones were taken for fur-
ther analysis: pCas1–3, pCat4–5, pMas6–8, pMel9–12,