Occasional Papers

Museum of Texas Tech University

Number 223 13August 2003

Rapidly Evolving Repetitive DNAin a Karyotypically Mecaevolved
Genome: Factors That Affect Chromosomal Evolution

Robert D. Bradley, Roslyn Martinez, MaryMaltbie, Irene Tiemann-Boege,

Holly A. Wichman, and Robert J. Baker

Among some congeneric species of mammals,
one taxon exhibits chromosomal stasis, whereas the
other species possess a radically reorganized karyo¬
type (Baker and Bickham, 1980). This phenomenon
of radically reorganizing the G-banded pattern of the
karyotype is termed karyotypic megaevolution and it
involves an extensive amount of chromosomal evolu¬
tion through the incorporation of both the numbers
and kinds and types of rearrangements. The most clas¬
sical example of karyotypic megaevolution is found in
two species of Muntiacus where diploid numbers are
2n= 6 for M. muntjak and 2n - 46 for M. re eves i
(Fredga, 1977). A significant aspect of this phenom¬
enon is that the euchromatic regions of the genome
can be changed substantially while having little or no
affect ai the phenotype (morphologic) level, at least
not a sufficient amount of change to justify generic
distinction. Karyotypic megaevolution probably rep¬
resents the extreme condition for rate of karyotypic
change involving complex chromosomal rearrange¬
ments (i.e. tandem fusions, peri and paracentric inver¬
sions). Relative to understanding the factors that af¬
fect chromosomal evolution, karyotypic megaevolution
provides examples for studies involving two closely
related species where one maintains chromosomal sta¬

sis while the other undergoes numerous chromosomal
rearrangements.

Many different factors have been proposed to
drive chromosomal evolution. These include demo¬
graphic models (Wright, 1941; Wilson et al., 1975;
Bush et al., 1977; Lande, 1979) and genetic and mo¬
lecular factors (Pathak et al,, 1973; Hsu et al., 1975;
Hatch et al., 1976; Finnegan et al., 1982; Shaw et al.,
1983; Wurster-Hill et al., 1988; Graphodatsky, 1989;
Baker and Wichman, 1990; Meyne et al., 1990; Redi et
al., 1990; Wichman et al., 1991). Of these hypoth¬
eses, only the tandem-repeat model by Wichman et al.
(1991) establishes a set of testable predictions. Spe¬
cifically, Wichman et al. (1991) proposed that lineages
undergoing rapid karyotypic change would have mul¬
tiple families of tandem repe ats; whereas lineages char¬
acterized by karyotypic stasis would have fewer fami¬
lies and a lower abundance of tandem repeats. In ad¬
dition, taxa possessing multiple repeats would be ex¬
pected to have these repeats actively changing chro¬
mosomal fields, as suggested by Lima-De-Faria (1980),
whereas in taxa expressing karyotypic stasis, these
repeats would be restricted to a single chromosomal
field. From a population genetics standpoint, litter-

2

Occasional Papers, Museum of Texas Tech University

size, effective population size, and generation time (i.e,
demographic and populational characteristics) do not
vary so radically in congeneric taxa that they could
explain the extreme differences in rates and types of
chromosomal rearrangements observed in karyotypic
megaevolution.

The tandem-repeat model (Wichman et al., 1991)
was based on studies of genome organization in equids.
Equids have been proposed to be the most rapidly evolv¬
ing karyotypic group of mammals (Wilson etal,, 1975).
Within the genome of six species of equids, it was
found that families of tandem repeats were diverse
and that the chromosomal fields (Lima-De-Faria, 1980)
occupied by these repeats varied substantially among
closely related species.

Using bats as a model, Bradley and Wichman
(1994) tested the hypothesis that chromosomal evolu¬
tion was associated with the occurrence of tandemly
repeated DNA sequences. The phylogenetic screen¬
ing method (Wichman et al. 1985, 1990) was used to
evaluate the activity of tandemly repeated sequences
in a conservatively evolving genome represented by
the bat species Macrotus waterhousii compared to the
activity of tandem repeats in the more rapidly evolving
genome of the outgroup taxon {Artibeus jamaicensis).
Their findings indicated that the number of families of
repeated sequences were lower in M. waterhousii than
in A Jamaicensis; thus providing initial support for the
Wichman et al. (1991) predictions concerning karyo¬
typic evolution. However, the study by Bradley and
Wichman (1994) represented only one end of the com¬
parative spectrum. What was lacking was an evalua¬

tion of the families and abundance of tandemly evolv¬
ing repeats from a taxon with a radically reorganized
karyotype (megaevolved) compared to a more con¬
servatively evolving karyotype (chromosomal stasis).

In this study, tandemly repeated DNA sequences
were examined from the bat species Rhinophylla
pumilio . This species was selected for two reasons.
First, if. pumilio possesses a radically evolved karyo¬
type having accumulated > 20 rearrangements com¬
pared to its sister taxon R, fischerae, This extensive
reorganization makes it impossible to compare to the
G-banded karyotype of if. pumilio to the proposed
primitive karyotype (Al. waterhousii) for the family
Phyllostomidae (Baker, 1979; Baker et al., 1989). On
the other hand, if. fischerae has a karyotype that dif¬
fers from the primitive condition by seven rearrange¬
ments: four fusions, one inversion, and two terminal
translocations (Baker et al. 1987). This characteristic
suggests that if. pumilio possesses one of, if not the
most evolved karyotype in the family Phyllostomidae,
Second, if. pumilio belongs to the same family
(Phyllostomidae) as A Jamaicensis and M, waterhousii
allowing for a comparison of tandemly repeated se¬
quences in a known phylogenetic framework. Spe¬
cifically, our goal was to compare the number of rap¬
idly evolving tandemly repeated DNA sequences found
in a species undergoing karyotypic megaevolution (if,
pumilio) to that found in a taxon demonstrating chro¬
mosomal stasis (M. waterhousii ). If the number of
tandemly repeated sequences in if. pumilio statistically
exceeds that found in M. waterhousii, then the hy¬
pothesis of Wichman et al. (1991) remains viable.

Methods and Materials

High molecular weight DNA was isolated from Sau3Al. Digests were electrophoresed on low melt-

approximately 0.5 g of liver tissue of A. jamaicensis ing point agarose gels. DNA fragments in the 4-6 kb

(TK 32042, male, Cuba: Guantanoma Province; range were extracted, ligated into the BamRl site of

Guantanoma Bay Naval Base) and if. pumilio (TK pUC 18 vector, and transformed by electroporation

17565, female, Surinam: Marowijne; 3 km SWAlbina). into the JM103 strain of E. colt
Methods for DNA isolation, cloning, digestion, trans¬
fer, and hybridization followed that of Bradley and Operationally, clones were not amplified during

Wichman (1994). Specifically, a genomic library was the transformation pro cess; thus each clone represented

constructed from if, pumilio by generating partial di- a unique DNA fragment and was treated as such by

gests of genomic DNA using the restriction enzyme assigning each an identification number. DNA from

Bradley et al.— Factors that Affect Chromosomal Evolution

3

each clone was triple digested with EcoRl, Hindlll ,
and Bam HI, electrophoresed on 0.8% agarose gels,
and DNA transferred to two positively charged nylon
membranes (Boehringer Mannheim) by placing filters
above and below the gel following a modified tech¬
nique of Southern (1975).

This generated two identical membranes each
with bound DNA from R. pumilio clones that were
used for the phylogenetic screening procedure
(Wichman et al., 1985). Each filter was hybridized to
labeled genomic probes, one from R. pumilio (ingroup)
and the other from A. jamaicensis (outgroup). Probes
were constructed from genomic DNA labeled with
[ 32 P]dCTP using random-primed labeling techniques.
Hybridization conditions were standardized with those
of Bradley and Wichman (1994) to provide a compari¬
son among bat species, and to compare rates obtained
from rodents (Wichman et al., 1990) and primates
(Lloyd et al., 1987).

A second verification was performed, hybridiz¬
ing the same membranes using the ECL Direct kit
(Amersham). Membranes were washed previous to
the hybridization in lx SSC, 1%SDS at 65°C for one
hour and rinsed two times in 2xSSC for five minutes
at room temperature. Genomic DNA of A. jamaicensis
and R . pumilio was sonicated and 1.5 pg were la¬
beled. DNA was hybridized at 42°C overnight in 150
ml hybridization solution (ECL kit). Post-hybridiza¬
tion washes included two washes in 2xSSC for five
minutes at room temperature, two washes in ECL post
hybridization solution (6M urea, 04% SDS, 0.5xSSC)
at 42°C for 10 minutes, and two washes in 2xSSC for

five minutes. Membranes were blotted and submerged
in reagents I and II for one minute and exposed on
film.

Autoradiograms of the two sets of filters (from
both experiments) were overlaid and compared for ab¬
sence or presence of hybridization of R . pumilio or A.
jamaicensis genomic DNA with the done fragments.
In addition, the difference in intensity was compared
by scoring bands as faint, medium, or strong. Under
these conditions, only repetitive sequences show de¬
tectable hybridization because single copy sequences
are under-represented in a total genomic probe. The
presence or absence of a band indicates the gain or
loss of a repetitive element or portion of the element,
whereas the difference in intensity indicates the num¬
ber of copies of that element.

Clones identified as different between the ingroup
and outgroup were verified by repeating the phyloge¬
netic screening procedure and were designated as
hypervariable. Hypervariable clones were sorted into
families by Southern blot cross-hybridization (South¬
ern, 1975). If clones possessed multiple bands (frag¬
ments), each band was numbered sequentially begin¬
ning with the largest fragment. Cross-hybridization
experiments involved labeling the hypervariable band(s)
of each clone and using it to probe the other
hypervariable clones. The bands of interest were ex¬
cised from 0.8% agarose gels, extracted and purified
with the Prep-A-Gene kit (Bio-Rad), radiolabeled, hy¬
bridized, and scored as described above. These ex¬
periments were repeated until all clones were assigned
to at least one family.

Results

Phylogenetic screening methods (Wichman et al.,
1985) were used to screen 747 clones from a genomic
library constructed from J?. pumilio DNA. Of these,
103 did not show any detectable hybridization to A.
jamaicensis or self genomic DNA, indicating that they
probably represented single or low copy sequences.
The average insert size per clone was approximately
4.9 kb. If bats possess a genome size that is approxi¬
mately 60-80% of the typical mammalian genome
(Manfredi-Romanini, 1985; Burton et al., 1989), then

the 747 clones examined represent approximately 1/
1000 of the bat genome. Two clones (Rp59 and Rp435)
were identified as hypervariable, in that they were
present in the ingroup (R, pumilio) but absent in the
outgroup (A. jamaicensis). The low intensity of hy¬
bridization of these two clones suggests that they are
members of a family of repeats characterized by low
copy number. Four additional clones (Rp418, Rp607,
Rp612, and Rp627) showed more intense hybridiza¬
tion in R. pumilio than in A, jamaicensis hut were not

4

Occasional Papers, Museum of Texas Tech University

considered as representing hypervariable clones as
these sequences would not be expected to contribute
to the hypotheses as outlined in Wichman et al. (1990).

The potential intenrelationship (sequence similar¬
ity) of these two clones was examined using cross¬

hybridization experiments. For each clone, bands (DNA
fragment) generated by the triple digest of BamH\,
£coRI, and FfmdIII were used as probes (Fig. 1). In
each case, the labeled fragment hybridized to itself and
other fragments from that clone; however, no frag¬
ment cross-hybridized to any other clone.

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Figure 1. Autoradiogram depicting the phylogenetic screening process for representative clones (DNA se¬
quences) isolated from a library constructed from genomic DNA obtained from Rhinophylla pumilio. Clones
were triple digested with-Sa/wHI, EcoKL, and Hindi II restriction enzymes and were then hybridized to a radioac-
tively-labeled probe constructed from total genomic DNA from Rhinophylla pumilio (left) and Artibeus
jamaicensis (right). Only hybridization to repetitive elements is visible under these conditions.

Discussion

Seven hundred forty seven inserts were exam¬
ined in this study. Of these, 103 did not show detect¬
able hybridization to total genomic DNA and thus are
thought to contain single or low copy sequences; con¬
sequently, they were removed from further consider¬
ation. The majority of the remaining clones (638 of
644) contained repetitive sequences that do not appear
to show different patterns of evolution since the di¬
vergence of R . pumilio and A. jamaicensis . Of the

remaining six clones, only two meet the criteria of
hypervariable as defined by Wichman et al. (1991).
These two DNA sequences appear to have originated
since R. pumilio and A. jamaicensis shared a common
ancestor. In addition, these clones appear to represent
different DNA sequence families, as they share no ap¬
parent sequence similarity in cross-hybridization ex¬
periments. Alternatively, these sequences could con¬
tain portions of a larger repeat.

Bradley et al.— Factors that Affect Chromosomal Evolution

5

Results of this study (Table 1) were similar to
that obtained from the phylogenetic screening of the
Macrotus genome (Bradley and Wichman, 1994), Both
genomes contained a paucity of hypervariable clones
and families of hypervariable sequences. However,

these data differed substantially from similar studies
of rodents (Wichman et al., 1985; 1990), primates
(Lloyd et al., 1987), and equids (Wichman et al., 1990;
1991).

Table 1. Comparison of results from phylogenetic screening efforts of rodents (Wichman et
al, 1985; 1990), equids (Wichman et al, 1990; 1991), primates (Lloyd et al, 1987), and
bats (Bradley and Wichman, 1994; this study).

Taxon

% Genome
Examined

# Variable
Clones

# Variable
Families

Average Genome
Size of Order (Mb)

Peromyscus

0.1

11

4

3400

Equus

0.1

34

6

3019

Homo

0.17

20

3

3000

Macrotus

0.1

I

1

1890

Rhinophylla

0.1

2

2

1890

Interpretation of the data generated from this
study require the rejection of the prediction by Wichman
et al. (1991) that lineages that undergo rapid karyo¬
typic change would have multiple families of tandemly
repeated sequences. However in rejecting this hypoth¬
esis, three alternative scenarios must be examined.
First, it may be that bats possess fewer repetitive se¬
quences than do other mammals. This possibility is
supported by the observation of Manfredi Romanini
(1985) and Burton et al, (1989) that bats possess ap¬
proximately 60-80% of the DNA found in typical mam¬
malian species. Although, the hypothesis that genome
size is proportional to the number of repeats or repeat
activity has been debated recently (Mouse Genome
Sequencing Consortium, 2002).

Second, our phylogenetic screening procedure
may not have detected the presence of some
hypervariable sequences. In this study, we choose A.
jamaicensis as the outgroup taxon for evaluating the
evolution of genomic sequences in the rapidly evolv¬
ing genome of R, pumilio, It may be that the genome
of A, jamaicensis contains many of the potentially rap¬
idly evolving sequences found in the R. pumilio ge¬
nome. It is known that A. jamaicensis possesses a
moderately evolving genome as calculated from the
accumulation of chromosomal rearrangements since

its divergence from the base of the Phyllostomid clade
(Baker et al., 1989). Additionally, it may be that the
ingroup taxon is too closely related to the outgroup
taxon. What may be more valuable is a comparison of
Rhinophytla to Macrotus. This would provide for a
more conservative test.

Third, for bats, it may be that tandemly repeated
sequences do not drive chromosomal evolution as is
hypothesized for equids (Wichman et al., 1990,1991).
Alternatively, some other molecular mechanism is re¬
sponsible for this pattern of karyotypic megaevolution.
For example, the rate of insertions/deletions or substi¬
tutions is very high in repeats of chiropterans and thus
these divergent families of repeats would be missed
by our stringent hybridization conditions. The com¬
parison of the mouse and human genome showed that
substitution rates among repeats vary significantly. Of
course, the longer the repeat the less the effect of the
substitution rate on the hybridization. Sequencing the
hypervariable clones would give us an indication of
the type and length of the repeat (Mouse Genome Se¬
quencing Consortium, 2002). Another possibility is that
sequences with a large number of repeats are difficult
to clone. The selection of 4-6kb fragment sizes could
have also contributed to the loss of repeat sequences.

6

Occasional Papers, Museum of Texas Tech University

Acknowledgments

We thank F. Mendez-Harclerode, S. Reeder, B.
Amman, and M, Haynie for reviewing earlier versions
of this manuscript. Thanks to B. Walker for assis¬
tance in the laboratory. Tissue samples were kindly
provided by the Natural Science Research Laboratory,
Museum of Texas Tech University. Support for this
research was obtained from the Albert R. and Alma

Shadle Fellowship, American Society of Mammalo-
gists (RDB), Howard Hughes Medical Institute, Un¬
dergraduate Biological Sciences Education Program to
Texas Tech University (RM), NIH Grant GM 38727
(HAW), NSF Grants BSR-86-00646 and BSR-90-
06797 (RJB).

Literature Cited

Baker, R. J. 1979. Karyology, Pp. 107-156 in Biology of
bats of the New World family Phyllostomidae,
Part ID (R. J. Baker, J. K. Jones Jr., and D, C. Carter,
eds.). Special Publications, The Museum, Texas
Tech University, Lubbock, Texas.

Baker, R, J., and J. W. Bickham. 1980. Karyotypic evolu¬
tion in bats: evidence of extensive and conser¬
vative chromosomal evolution in closely related
taxa. Systematic Zoology, 29:239-251,

Baker, R. J., and H. A. Wichman. 1990. Retrotransposon
Mys is concentrated on the sex chromosomes:
implications for copy number containment. Evo¬
lution, 44:2083-2088.

Baker, R. J., C. S, Hood, and R, L. Honeycutt, 1989. Phylo¬
genetic relationships and classification of the
higher categories of the New World bat family
Phyllostomidae, Systematic Zoology, 3 8:228-23 8.

Baker, R. J., M, B. Qumsiyeh, and C. S, Hood, 1987. Role
of chromosomal banding patterns in understand¬
ing mammalian evolution. Pp. 67-96 in Current
Mammalogy, Vol. 1 (H, H. Genoways, ed,). Ple¬
num Publishing Corporation, New York.

Bradley, R. D,, and H. A. Wichman. 1994. Rapidly evolv¬
ing repetitive DNAs in a conservative genome: a
test of factors that affect chromosomal evolu¬
tion. Chromosome Research, 2:354-360,

Burton, D. W., J. W. Bickham, and H. H. Genoways. 1989.
Flow cytometric analyses of nuclear DNA in four
families of neotropical bats. Evolution, 43:756-
765.

Bush, G. L,, S. M, Case, A. C. Wilson, and J, L. Patton.
1977. Rapid speciation and chromosomal evolu¬
tion in mammals. Proceedings of the National
Academy of Science, 74:3942-3946.

Finnegan, D. J., B. H. Will, A, A. Bayev, A. M. Bowcock,
and L. Brown. 1982. Transposable DNA se¬
quences in eukaryotes. Pp. 29-40 in Genome
Evolution (G. A. Dover, and R. B. Flavell, eds,).
Academic Press, London.

Fredga, K. 1977. Chromosomal changes in vertebrate
evolution. Proceedings of the Royal Society of
London, 199:377-397.

Graphodatsky, A. S. 1989. Conserved and variable ele¬
ments of mammalian chromosomes. Pp, 95-123
in Cytogenetics of animals (C. R. E. Halhan, ed.).
CAB International Press, Oxon, United Kingdom.

Hatch, F, T., A. J. Bodner, J, A. Mazrimas, and D. H. Moore.
1976, Satellite DNA and cytogenetic evolution:
DNA quantity, satellite DNA, and karyotypic
variation in kangaroo rats (genus Dipodomys).
Chromosoma, 58:155-168.

Hsu, T. S. Pathak, and T. R. Chen. 1975. The possibility
of latent chromosomes and a proposed nomen¬
clature system for total chromosome and whole
arm translocations. Cytogenetics and Cell Ge¬
netics, 15:41-49.

Lande, R, 1979. Effective deme size during longterm evo¬
lution estimated from rates of chromosomal evo¬
lution. Evolution,33:234-251.

Lima-De-Faria, A. 1980. Classification of genes, rearrange¬
ments and chromosomes according to the chro¬
mosome field, Hereditas, 93:1-46.

Lloyd, J. A., A. N, Lamb, and S. S. Potter. 1987. Phyloge¬
netic screening of the human genome: identifica¬
tion of evolutionarily variable repetitive sequence
families. Molecular Biology and Evolution, 4:85-
98.

Bradley et al.—Factors that Affect Chromosomal Evolution

7

Manfredi-Romanini, M. G 1985. The nuclear content of
deoxyribonucleic acid and some problems of
mammalian phylogenesis. Mammalia, 49:369-385.

Meyne, J. 5 R. J. Baker, H. H. Hebarf, T. C. Hsu, O. A. Ryder,
O. G. Ward, J. E. Wiley, D. H. Wuster-Hill, T. L.
Yates, and R. K. Moyzis. 1990. Distribution of
nontelomeric sites of the (TTAGGG)^ telomeric
sequence in vertebrate chromosomes.
Chromosoma, 99:3-10.

Mouse Genome Sequencing Consortium. 2002. Initial
sequencing and comparative analysis of the
mouse genome. Nature, 420:520-562.

Pathak, S., T, C Hsu, and F E. Arrighi. 1973. Chromo¬
somes of Peromyscus (Rodentia, Cricetidae): IV.
The role of heterochromatin in karyotypic evolu¬
tion. Cytogenetics and Cell Genetics, 11:315-326.

Redi, C. A., S. Garagna, and M. Zuccotti. 1990.
Robertsonian chromosome formation and fixa¬
tion: the genomic scenario. Biological Journal
Linnean Society, 41:23 5-255.

Shaw, D. D., P. Wilkinson, and D. J. Coates. 1983. In¬
creased chromosomal mutation rate after hybrid¬
ization between two species of grasshoppers.
Science, 220:1165-1167.

Southern, E. M. 1975. Detection of specific sequences
among DNA fragments separated by gel electro¬
phoresis. Journal of Molecular Biology, 98:503-
517.

Addresses of authors:

Robert D. Bradley

Department of Biological Sciences and Museum
Texas Tech University
Lubbock, Texas 79409-3131
e-mail: robert.bradley@ttu.edu

Roslyn Martinez

Wichman, H, A., C. T. Payne, and T. W. Reeder. 1990.
Intrageneric variation in repetitive sequences iso¬
lated by phylogenetic screening of mammalian
genomes, Pp. 153-160 /k Molecular Evolution
(M. Clegg and S. J. O’Brien, eds.). Alan R. Liss
Inc., New York.

Wichman, H. A., S. S, Potter, and D. S. Pine. 1985. Mys, a
family of mammalian transposable elements iso¬
lated by a phylogenetic screening procedure. Na¬
ture, 317:77-81.

Wichman, H. A., C. T. Payne, O. A. Ryder, M. J. Hamilton,
M, Maltbie, and R. J. Baker. 1991. Genomic dis¬
tribution of heterochromatin sequences in
equids: implications to rapid chromosomal evo-
1 uti on. Joum al of Heredity, 82:369-377.

Wilson, A. C., G. L. Bush, S. M, Case, and M. C, King.

1975. Social structuring of mammalian popula¬
tions and rate of chromosomal evolution. Pro¬
ceedings of the National Academy of Science,
72:5061-5065.

Wright, S. 1941. On the probability of fixation of recipro¬
cal translocations, American Naturalist, 75:513-
525.

Wurster-Hill, D. H., O. G Ward, B. H, Davis, J. P. Park, R. P.

Moyzis, J. Meyne. 1988. Fragile sites, telomeric
DNA sequences, B chromosomes, and DNA con¬
tent in raccoon dogs, Nyctereutes procyonides ,
with comparative notes on foxes, coyotes, wolf
and raccoon. Cytogenetics and Cell Genetics,
49:278-281.

Mary Maltbie

Los Alamos National Laboratory

Life Sciencse Division

Mail Stop M880

Los Alamos ; New Mexico 87545

Current Address:

Therion International, LLC
36 Phila Street

Saratoga Springs, New York 12866
e-mail: maltbie@theriondnaxom

Department of Biological Sciences
Texas Tech University
Lubbock, Texas 79409-3131

Current Address:

8023 Garden Court
San Antonio, Texas 78239
e-mail: roslyn76@flash.net

8

Occasional Papers, Museum of Texas Tech University

Irene Tiemann-Boege

Department of Biological Sciences
Texas Tech University
Lubbock, Texas 79409-3131

Current Address:

Molecular and Computational Biology Program
University of Southern California
Los Angeles , California 90089-1340
e-mail; tiemanbo@usc.edu

Holly A. Wichman

Department of Biological Sciences
University of Idaho
Moscow, Idaho 83843
e-mail: hwichman@uidaho.edu

Robert J. Baker

Department of Biological Sciences and Museum
Texas Tech University
Lubbock, Texas 79409-3131
e-mail: rjbaker@ttu.edu

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