Syst. Biol. 46(4)579-589, 1997
DNA SYNAPOMORPHIES FOR A VARIETY OF TAXONOMIC LEVELS
FROM A COSMID LIBRARY FROM THE NEW WORLD BAT
MACROTUS WATERHOUSII
ROBERT J. BAKER, 1 JONATHAN L. LONGMIRE, 2 MARY MALTBIE, 1
MEREDITH J. HAMILTON, 1 - 3 AND RONALD A. V A N D E N BUSSCHE 1 ' 3
department of Biological Sciences and The Museum, Texas Tech University, Lubbock, Texas 79409, USA;
E-mail: [email protected] (R.J.B.)
Genomics Group, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA
2
Abstract.—An effective method yielding taxon-specific markers from the genome of a single individual would be valuable for many types of scientific investigations, including systematic, forensic, conservation, and evolutionary studies. We explored the use of cosmid libraries, with insert
sizes averaging 35 kb, to streamline the process of locating sequences of DNA that can serve as
taxonomic markers from the specific to the ordinal levels. By screening approximately 2.6% of the
leaf-nosed bat (Macrotus waterhousii) genome, we identified several potential DNA fragments that
appear to be synapomorphic for a variety of taxonomic levels. A more thorough analysis of the
markers documented that 17 Macrotws-specific clones represent three distinct DNA generic markers, whereas 30 microchiropteran clones represent multiple copies of a single family of repetitive
DNA. The Microchiroptera taxon markers hybridize with representatives of most of the Microchiroptera families; however, no hybridization was detected for members of the superfamily Rhinolophoidea. These results demonstrate that cosmid libraries can be a valuable source for isolating
taxon-specific markers from mammals even when the insert size is as large as 35 kb. [Cosmid
library; DNA synapomorphies; genome; Macrotus waterhousii; phylogenetic screening; Rhinolophoidea; taxonomic levels.]
Genomes of more complex organisms
are composed of billions of base pairs that
contain sequences that range from being
unique for individuals to some, such as ribosomal genes, that show considerable
similarity among all forms of life (Gouy
and Li, 1989). Although there is considerable potential for finding DNA markers
that resolve all taxonomic levels, the problem is how to extract efficiently the desired
levels of variation and shared character
states from these billions of base pairs. An
effective method that yields potential taxon-specific DNA probes across taxonomic
levels would be valuable for many types of
scientific investigations, including systematic, forensic, conservation, and evolutionary studies. We explored the use of cosmid
libraries to streamline the process of locating sequences of DNA that can serve as
3
Present address: Department of Zoology, Life
Sciences West, Oklahoma State University, Stillwater,
Oklahoma 74078, USA; E-mail: [email protected].
edu (M.J.H.), [email protected]
(R.V.D.B.).
taxonomic markers from the ordinal to the
specific level.
We tested our approach by constructing
a genomic cosmid library from the New
World leaf-nosed bat, Macrotus waterhousii,
a species whose specific, generic, familial,
superfamilial, subordinal, and ordinal relationships are well resolved (Baker et al.,
1989; Van Den Bussche, 1991; Koopman,
1993). Although there has been recent debate over the monophyly of the order Chiroptera (Baker et al., 1991a, 1991b; Pettigrew, 1991a, 1991b; Simmons et al., 1991),
nearly all data, with the possible exception
of some neural characters of the brain (Pettigrew, 1986) support the monophyly of
Chiroptera (for review, see Simmons, 1994;
Van Den Bussche et al., unpubl.). One assumption of our experimental design is
that the groups in Figure 1 are monophyletic. It was our goal to locate clones that
defined the taxonomic limits of the genus
Macrotus, the family Phyllostomidae, the
superfamily Noctilionoidea, the suborder
Microchiroptera, and the order Chiroptera.
We examined 1,728 independent clones,
579
580
SYSTEMATIC BIOLOGY
VOL. 46
from Cuba (Guantanamo Prov., Guantanamo Bay Naval Base, TK 32184). The liJ? /
(7 clones)
brary was constructed using the same
methods as those used to construct human
chromosome-specific cosmid libraries
(Longmire et al., 1993). The characteristics
of this library have been described (Van
Macrotus (17 clones)
Den Bussche et al., 1995). In general, priPhyllostomidae (10 clones)
mary infection of Escherichia coli host strain
DHaMCR yielded 7.4 X 105 independent
Noctilionoidea (7 clones)
recombinants. From this primary infection,
Microchiroptera (44 clones)
1,728 independent clones were chosen,
—|— Noctili
Chiroptera
(31 clones)
grown, and archived into 96-well microtiter plates. A replica plater (Sigma Chemi_ Microchiroi
FIGURE 1. _ Phylogenetic
relationships of the higher cal Co.) was used to inoculate nylon memtaxonomic categories
tested
Chiroptera
(31for the isolation of taxonspecific markers isolated from a cosmid library of ge- branes (Biodyne B 0.45 jxm) with clones
nomic DNA from Macrotus waterhousii. The number of from the microtiter plates. Membranes
potential taxon-specific clones isolated from the M. were incubated at 37°C for 7 hr on LB agar
waterhousii library is given for each taxonomic cate- containing kanamycin (30 mg/ml) and
gory.
then transferred and incubated overnight
at 37°C on LB agar containing kanamycin
with an average insert size of approxi- and chloramphenicol (170 mg/ml; Sammately 35 kb, by probing with genomic brook et al., 1989). DNA was fixed by placDNA from 15 taxa to isolate clones that ing the membranes sequentially on blothave potential to provide taxon-specific ting pads soaked in 0.4 M NaOH (5 min),
resolution. In a recent study (Van Den 0.5 M Tris/1.5 M NaCl, pH 7.5 (5 min),
Bussche et al., 1995), these 1,728 clones and 2x sodium citrate-sodium chloride
were characterized for the presence of five (SSC) (5 min), followed by baking at 80°C
repetitive elements known to be ubiqui- for 2 hr.
tous in the mammalian genome. These
Isolation of Taxon-Specific Markers
data will aid in understanding the nature
of the clones that identify taxonomic limits
Genomic DNAs were isolated from six
so that this potential source of noise in the representatives of the microchiropteran
data can be eliminated.
family Phyllostomidae (Macrotus water-
w
MATERIALS AND METHODS
We first constructed a genomic library
and plated random clones in an ordered
arrangement for archival purposes and for
hybridization. We then hybridized clones
with genomic DNA probes from individuals representing a subset of each taxon
(ingroups) and a subset of the outgroups.
Clones that hybridized with all ingroup
representatives for each taxon but not with
the outgroups for that taxon were further
examined as putative taxon-specific markers.
housii, M. californicus, Desmodus rotundas,
Micronycteris hirsuta, Phyllostomus elongatus,
Artibeus jamaicensis), five additional micro-
chiropteran families (Noctilionidae, Mormoopidae, Emballonuridae, Molossidae,
Vespertilionidae), one representative of the
suborder Megachiroptera (Pteropodidae),
and representatives of the orders Dermoptera, Insectivora, and Primates. Tissues
were obtained from the frozen tissue collection at The Museum at Texas Tech University. DNA isolation was performed using a modification of the technique of
Longmire et al. (1991). (See the Appendix
Construction and Characterization of the
for a list of specimens used in various
Cosmid Genomic Library
parts of this study.)
One microgram of each DNA sample
High-molecular-weight DNA was isolated from a male M. waterhousii collected was nick translated and used to probe the
1997
BAKER ET AL.—SOLATION OF DNA TAXON MARKERS
M. waterhousii library. Following hybridization, the 1,728 clones were scored on a
scale of 0 (=no detectable hybridization) to
3 (=a completely black spot on the autoradiograph for that clone; maximum detectable hybridization). Prior to hybridization, membranes were washed for 1 hr at
65°C in 0.1 X SSC, 0.1% sodium dodecyl
sulfate (SDS). Prehybridization was carried
out at 65°C for 1 hr in 6X SSC, 40% formamide (Kodak), 1% SDS, 0.005 M ethylenediaminetetraacetic acid (pH 8.0), and
0.005 g/ml powdered milk. Membranes
were hybridized overnight at 42°C in fresh
prehybridization solution containing approximately 1 X 10 6 cpm/ml probe. Probes
were labeled with a (a32P) dCTP by nick
translation; the nonincorporated label was
removed by spin column chromatography
(Sambrook et. al, 1989). Prior to hybridization, probes were denatured for 10 min at
37°C in 0.1 M NaOH. Following hybridization, membranes were washed once for 15
min in 2X SSC, 0.1% SDS at room temperature and twice for 15 min in 0.1 X SSC,
0.1% SDS at 50°C. Washed membranes
were exposed at -80°C to Kodak XAR-5
film and two lighting plus intensifying
screens.
Once putative taxon-specific clones were
identified for each taxonomic level, we
eliminated any clones that were identified
by Van Den Bussche et al. (1995) as having
tandemly repeated elements (microsatellites, etc.) that are ubiquitous among genomes. A series of Southern blot analyses
(Southern, 1975) were performed to determine the complexity of collections of
clones that were informative at either the
generic or subordinal levels. Cosmid miniprep DNA was digested with EcoRI and
electrophoresed on 0.8% agarose gels. The
gels were blotted to nylon hybridization
membranes and hybridized with a single
clone from the informative collection. Any
clone that produced detectable cross-hybridization with the first chosen informative clone (clone that was used as a probe)
was classified as belonging to the same
family of repetitive DNA. DNA from all
other clones (those that did not cross-hybridize with the specific clone used as a
581
probe) were used in a subsequent
Southern blot analysis, and an arbitrarily
chosen representative of this group was
used to probe this membrane. As with the
initial survey, all clones producing detectable hybridization were classified as belonging to a second family of repetitive
DNA. This process was repeated until all
clones were assigned to a specific family of
repetitive DNA based on cross-hybridization experiments.
To evaluate the robustness of the putative taxonomic markers, a representative of
the Macrotus- and Microchiroptera-specific
clones were used as probes against slot
blots of genomic DNA. In these experiments, we increased the phylogenetic
breadth of the survey by including representatives of 21 genera of the family Phyllostomidae, all three families of the superfamily Noctilionoidea, eight families of the
suborder Microchiroptera, five genera of
the suborder Megachiroptera, one species
of Dermoptera, two families of the order
Primates, and one genus of each of the orders Rodentia and Insectivora. One microgram of genomic DNA from each of the
above taxa was applied to hybridization
membranes following the manufacturer's
recommended procedure (Schleicher &
Schuell, Keene, NH). Hybridization conditions were the same as those used in the
first part of the experiment except that various stringency levels were explored, with
hybridization conditions ranging from
40% formamide and 42°C to 50% formamide and 65°C. Based on the hybridization
conditions, appropriate adjustments were
made in posthybridization washes by
varying both the temperature and salt concentration.
In situ Hybridization
Chromosomal preparations from M. waterhousii, M. californicus, and Artibeus ja-
maicensis were prepared from bone marrow after incubation with Velban (Baker
and Qumsiyeh, 1988). In situ hybridization
of a representative cosmid of the Macrotusand Microchiroptera-specific repetitive
DNA families was performed according to
the method of Hamilton et al. (1990). A
582
VOL. 46
SYSTEMATIC BIOLOGY
TABLE 1. Total number and percentage of the 1,726 a density that allowed detection of the coscosmids from the Macrotus waterhousii library produc- mid DNA with a standard miniprep proing detectable hybridization with genomic DNA from
cedure, reducing the actual number of cos15 mammalian taxa.
No.
Taxon
positive
cosmids
% cosmids
hybridizing
1,559
1,493
1,609
1,521
1,418
1,176
90
86
93
88
82
66
1,301
75
991
57
885
51
1,329
77
955
55
810
47
475
27
521
30
882
51
Chiroptera
Microchiroptera
Noctilionoidea
Phyllostomidae
M. waterhousii
M. californicus
Desmodus
Micronycteris
Phyllostomus
Artibeus
Mormoopidae
Pteronotus
Noctilionidae
Noctilio
Emballonuridae
Saccopteryx
Molossidae
Tadarida
Vespertilionidae
Myotis
Megachiroptera
Pteropus
Dermoptera
Cynocephalus
Primates
Homo
Insectivora
Crocidura
minimum of 10 spreads were analyzed for
each individual examined.
RESULTS
Characterization of the M. waterhousii
Genomic Library
mids screened to 1,726. The remaining 33
clones were visible on an ethidium-stained
gel; when digested with EcoRI, each clone
was verified as having the 6.7-kb DNA
fragment characteristic of the sCos-1 vector
as well as additional bands resulting from
the insert DNA.
The insert size of 20 randomly selected
cosmid clones ranged from 24.9 to 43.4 kb,
with a mean insert size of 35.7 kb. Based
on this mean insert size, the 1,726 clones
represented 6.15 X 107 bp, or 2.6% of the
M. waterhousii genome, assuming a genome
size of 2.4 X 109 bp (based on a DNA content of 5.6 pg/cell; J. W. Bickham, pers.
comm.). The total library of 7.4 X 105 primary clones thus is 11-fold representative
for the M. waterhousii genome.
Taxon-Specific Clones
The hypothesized phylogeny for the
taxa used in this study along with the total
number of clones that are potential markers for each taxonomic category are presented in Figure 1. Based on cross-hybridization results, the 17 Macrotws-spetific
clones represent three families comprised
of 8, 7, and 2 clones, respectively. Of the
44 potential microchiropteran clones, 14
were eliminated because they contained
other micro- or minisatellite repeats (Van
Den Bussche et al., 1995). The remaining
30 clones cross-hybridized, indicating that
these clones represent multiple members
of a single family of repetitive DNA.
When clones representing each of the
three families of Mocrofws-specific DNA
were used to probe slot-blot membranes,
detectable hybridization was seen only for
The pattern of hybridization of genomic
DNA from the 15 mammalian taxa studied
is provided in Table 1. Only 35 of the 1,728
cosmid clones screened in this study did
not hybridize with any of the 21 (15 from M. waterhousii and M. californicus (Fig. 2).
the microchiropteran species plus 6 repet- This same pattern was detected regardless
itive elements examined by Van Den of the stringency of hybridization and
Bussche et alv 1995) probes examined. Of washes ranging from hybridization in 40%
these 35 clones (2.0% of those screened), 2 formamide at 42°C to hybridization in 50%
produced no bands when they were di- formamide at 65°C and posthybridization
gested with the restriction endonuclease washes in 0.1 X SSC at temperatures rangEcoRI and electrophoresed on an 0.8% aga- ing from 42°C to 65°C. Results from in situ
rose gel. These two clones apparently did hybridization demonstrated that these
not contain a cosmid or failed to grow to clones were localized in discrete blocks on
1997
BAKER ET AL.—SOLATION OF DNA TAXON MARKERS
M. waterhousii
^ ^ B
M. californicus
^fc
583
Noctilio
Pteronotus
Micronycteiis
Natalus
Desmodus
Furipterus
Diaemus
Rhinolophus
Vampyrum
Tadarida
Chrotopterus
Hipposideros
Phyllostomus
Megaderma
Glossophaga
Saccopteryx
Anoura
Nycteris
Choeroniscus
Macroglossus
Brachyphylla
Megaloglossus
Sturnira
Nyctimene
Hhinophylla
Pteropus
Centurio
Rousettus
Stenoderma
Cynocephalus
Artibeus
Lemur
Chiroderma
Homo
Vampyrodes
Scalopus
Vampyressa
Mus
Uroderma
FIGURE 2. Autoradiogram of a slot-blot analysis in
which 1 (xg of genomic DNA from each taxon was
transferred to hybridization membrane and then hybridized with one of the Macrofws-specific cosmids.
the chromosomes of M. waterhousii and M.
californicus (Figs. 3a, 3b). In the slot-blot experiments, the level of hybridization was
reduced in M. californicus compared with
that seen in M. waterhousii (Fig. 2), and the
lower abundance of these sequences in M.
californicus also was observed in the in situ
hybridization experiments. In M. waterhousii, this DNA hybridized with approximately 38 chromosomes at either one or
both telomeres. However, in M. californicus
hybridization was restricted to a single
telomeric region on two small chromosomes (Figs. 3a, 3b).
When a representative of the Microchi-
FIGURE 3. Representative karyotypes of three bat
species. The clones used as probes were labeled with
biotin. Regions of hybridization fluoresce yellow (fluorescein) and unhybridized regions of the chromosome
fluoresce red (propidium iodide), (a) Macrotus waterhousii, in situ hybridized with one of the Macrotusspecific clones, (b) Macrotus californicus, in situ hybridized with one of the Mocrofus-specific clones, (c) Artibeus jamaicensis, in situ hybridized with a representative of a Microchiroptera-specific clone.
roptera-specific family of 30 clones was hybridized with the slot-blot membranes, intense hybridization was detected for all
noctilionoid bats (Phyllostomidae, Nocti-
584
M. waterhousii
M. californicus
VOL. 46
SYSTEMATIC BIOLOGY
t
Noctilio
Pteronotus
Micronycteris
Natalus
Desmodus
Furipterus
Diaemus
Rhinolophus
Vampyrum
Tadarida
Chrotopterus
Hipposideros
Phyllostomus
Megaderma
Glossophaga
Saccopteryx
Anoura
Nycteris
Choeroniscus
Macroglossus
Brachyphylla
Megaloglossus
Sturnira
Nyctimene
Rhinophylla
Pteropus
Centurio
Rousettus
Stenoderma
Cynocephalus
Artibeus
Lemur
Chiroderma
Homo
Vampyrodes
Scalopus
Vampyressa
Mus
range of stringencies for hybridization and
posthybridization conditions. The only notable variation in these results was that as
the level of stringency was increased, the
intensity of hybridization became lower in
the nonphyllostomid microchiropterans. In
situ hybridization of a representative of
this family of DNA with the chromosomes
of Artibeus jamaicensis revealed an inter-
spersed pattern of hybridization on all
chromosomes, with some chromosomal
banding present (Fig. 3c).
DISCUSSION
Uroderma
FIGURE 4. Autoradiogram of a slot-blot analysis in
which 1 |xg of genomic DNA from each taxon was
transferred to hybridization membrane and then hybridized with one of the Microchiroptera-specific cosmids.
lionidae, Mormoopidae), whereas less intense hybridization was detected for representatives of the microchiropteran
families of Natalidae, Furipteridae, Molossidae, and Emballonuridae. Representatives of the microchiropteran families
Rhinolophidae, Hipposideridae, Megadermatidae, and Nycteridae produced no detectable hybridization. This clone also
failed to hybridize with any representatives of the Megachiroptera, Primates, Insectivora, or Rodentia (Fig. 4). As with the
Macrotus-specific clones, similar patterns of
hybridization were detected over the entire
Taxon-Specific Markers
Although satellite DNA has been shown
to be a useful phylogenetic marker for several taxa, including rodents (Hamilton et
al., 1990, 1992), bats (Van Den Bussche et
al., 1993), cetaceans (Arnason and Best,
1991; Arnason et al., 1992; Adegoke et al.,
1993), primates (Durfy and Willard, 1990),
falcons (Longmire et al., 1988), coral (McMillan and Miller, 1990; McMillan et al.,
1991), and Drosophila (Bachmann et al.,
1992), the isolation of these markers is usually time consuming and fortuitous. A
more efficient method that screens the genome with the specific purpose of isolating
taxon-specific markers would be valuable.
Because other researchers have isolated
stretches of DNA that are specific to species (Love and Deininger, 1992), subgenera
(Hamilton et al., 1990), and genera (Longmire et al., 1988; Hamilton et al., 1992; Van
Den Bussche et al., 1993), it should be possible to isolate, from a given genome, those
stretches of DNA.that identify that particular individual for a wide range of taxonomic groupings.
We have screened a cosmid library with
the specific goal of identifying marker sequences that would be useful for addressing taxonomic questions at several levels.
For all taxonomic categories examined,
several cosmids produced hybridization
patterns that were consistent with accepted monophyletic groupings (hybridized
with all ingroups but not to any outgroups). How many of the potential taxonomic markers are simply copies of the
1997
BAKER ET AL.—SOLATION OF DNA TAXON MARKERS
585
TABLE 2. Number of potential markers for taxo- 17 cosmids represent three unique families
nomic categories from species to superorder isolated of repetitive DNA; representatives of each
from the Macrotus waterhousii library and the number
and percentage of these potential taxon-specific mark- family do not cross-hybridize with representatives of the other two families, even
ers that hybridize with dinucleotide repeats.
under moderate levels of stringency for hybridization and posthybridization washes.
To test the efficiency of these three famTaxon
ilies of cosmids for identifying the genus
Macrotus (which contains only two spe1
M. waterhousii
6
17
cies), we hybridized these three probes
Macrotus
17
0
0
10
10
1
Phyllostomidae
with genomic DNA from 42 mammals rep7
29
2
Noctilionoidea
resenting 21 genera of phyllostomid bats,
44
32
14
Microchiroptera
11 microchiropteran families, and repre7
31
23
Chiroptera
sentatives of the Megachiroptera, Primates,
247
255
97
"Archonta"
Dermoptera, Rodentia, and Insectivora.
The potential Macrotus-speafic cosmids
same repetitive element or family of repet- hybridized only with the genomic DNA
itive elements, and how robust are these from M. waterhousii and M. californicus (Fig.
clones in resolving the proposed phyloge- 2). Thus, screening of 2.6% of the genome
netic limits? To test these two questions, of Macrotus resulted in isolation of three
we examined the clones for informative- unique genus-specific markers.
ness at the generic and subordinal levels.
Subordinal Level Markers
Because this library was used in a previous
In
contrast
to the Macrofws-specific
study to examine the organization of repetitive DNA in the M. waterhousii genome clones, in which three unique families of
(Van Den Bussche et al., 1995), we can repetitive DNA were isolated, all 30 Microidentify which of the potential taxon-spe- chiroptera-specific clones appear to belong
cific clones contain members of ubiquitous to the same family of interspersed repetirepeat families, such as microsatellites. tive DNA (Figs. 3c, 4). In situ hybridization
There was a general trend toward the of representatives of this family of repetihigher taxonomic levels for an increase in tive DNA with the chromosomes of Artipercentage of potential taxon-specific beus jamaicensis revealed that this family of
markers containing microsatellite clusters repeats is interspersed and present on all
(Table 2). These data point out one of the chromosomes, which may be why this redifficulties in isolating taxon-specific peat was present in 30 of our 1,726 clones.
markers. Microsatellite repeat clusters are Although in situ hybridization of this famubiquitous in vertebrate genomes (Tautz ily produced some chromosomal banding,
and Renz, 1984; Stallings et al., 1991; Beck- the banding pattern was not as discrete as
man and Weber, 1992; Janecek et al., 1993; seen with long interspersed repetitive eleVan Den Bussche et al., 1995) and therefore ments (LINEs) and short interspersed rewill hybridize across a broad array of taxa, petitive elements (SINEs) in humans and
giving false indication for taxon-specific deer mice (Korenberg and Rykowski, 1988;
boundaries that may not be the product of Baker and Wichman, 1990; Baker and Kass,
1994).
common ancestry.
Hybridization of this family of repetitive
Generic Level Markers
sequences with genomic DNAs from 42
Seventeen cosmid clones contained mammals demonstrates that these clones
stretches of DNA from the M. waterhousii hybridize only with representatives of the
genome that produced detectable levels of suborder Microchiroptera (Fig. 4). Howhybridization only with genomic DNA ever, under all conditions of hybridization,
from its sister taxon, M. californicus. Based representatives of the Old World microchion cross-hybridization experiments, these ropteran families Rhinolophidae, HipposiNo. cosmids
containing
No.
% micromicrocosmids
satellites satellites
586
SYSTEMATIC BIOLOGY
deridae, Megadermatidae, and Nycteridae
produced no detectable hybridization (Fig.
4). There are three possible explanations
for these results: (1) this DNA element in
members of the superfamily Rhinolophoidea has diverged considerably from that of
other microchiropteran taxa; (2) these four
taxa do not form a monophyletic group
within the other microchiropteran taxa; or
(3) some genomic mechanism has drastically reduced or eliminated copy number
of this family of repetitive DNA in these
taxa. The four families, Rhinolophidae,
Hipposideridae, Megadermatidae, and
Nycteridae, are recognized as belonging to
the microchiropteran superfamily Rhinolophoidea (Smith, 1976; Van Valen, 1979;
Koopman, 1984; Pierson, 1986). Higher taxonomic relationships within the Microchiroptera are not well resolved and are controversial (Baker et al., 1991a). However,
the absence of this repetitive element in
representatives of these four families may
document closer phylogenetic relationships of the remaining microchiropteran
families to the exclusion of the superfamily
Rhinolophoidea. Because elimination of a
group of complex repetitive elements from
a genome is more probable (especially if
they occur in discrete blocks of heterochromatin) than the de novo evolution of such
sequences, the presence of an element in a
genome may be viewed as strong evidence
for shared ancestry. However, at present it
is unclear how often such repetitive elements can be eliminated, giving a false
negative. The 30 clones isolated in this
study provide evidence for shared ancestry of those microbat families that are positive for the repeat. However, the absence
of this repetitive family in the rhinolophoids is more problematic. The observation that this family of repetitive sequences
is distributed in an interspersed fashion on
all chromosomes makes it less probable
that all or most copies could be eliminated.
We examined only 2.6% of the M. waterhousii genome to locate this marker. Screening of a larger representation of the Macrotus genome and the genome of other
microchiropteran bats may be successful
in isolating additional markers with phy-
VOL. 46
logenetic information among families of
Microchiroptera.
Previous molecular studies designed to
elucidate the phylogenetic relationships
among families of bats have failed to provide a robust number of synapomorphies
documenting sister-group relationships
(Baker et al., 1991a). The reason for this
lack of resolution may be that several families of bats shared a common ancestor for
a very short time before the lineages underwent a rapid radiation (Baker et al.,
1991a). Screening large-insert-size libraries
with genomic DNA from a taxonomically
diverse group of organisms may provide
resolution for higher taxonomic relationships by identifying large conservative
stretches of DNA. Nonetheless, repetitive
DNA has many problems for use in systematic studies because of the ubiquity of
many of these DNA families in all eukaryotic organisms. Moreover, although in
some DNA families all members share
identical sequence structure because of a
common evolutionary history and concerted evolution (Dover, 1982, 1986), members
of other DNA families are able to evolve
freely and the orthology of such fragments
can cause problems in phylogenetic analyses (Tautz and Renz, 1984; Burton et al.,
1986; Deininger and Daniels, 1986; Vassart
et al., 1987; Deininger and Slagel, 1988;
Deininger, 1989; Hutchison et al., 1989;
Moyzis et al., 1989; Durfy and Willard,
1990; Janecek et al., 1993; Lee et al., 1996).
We have demonstrated that phylogenetic
screening (Wichman et al., 1985) of a cosmid library with a broad taxonomic array
of genomic DNAs can find markers with
taxon-specific resolving power. However,
before undertaking such a study there are
issues to be considered, including the insert size to be employed in the construction of the library, the stringency used for
hybridization and posthybridization
washes, and the potential confounding effect that known families of repetitive DNA
such as microsatellites, LINEs, SINEs, and
various other transposable elements may
have on the identification and isolation of
taxon-specific markers. There are many
different kinds of DNA libraries that can
1997
BAKER ET AL.—SOLATION OF DNA TAXON MARKERS
587
be constructed from the genome of an in- ARNASON, U., AND P. B. BEST. 1991. Phylogenetic relationships within the Mysticeti (whalebone whales)
dividual. From the issues addressed here,
based upon studies of highly repetitive DNA in all
the size of the vector insert appears to be extant species. Hereditas 114:263-269.
the most important variable. The trade-off ARNASON, U., S. GRETARSDOTTIR, AND B. WIDEGREN.
is that the smaller the piece of DNA in- 1992. Mysticeti (baleen whale) relationships based
upon the sequence of the common cetacean DNA
serted into a cloning vector, the more
satellite. J. Mol. Evol. 9:1018-1028.
clones need to be screened to examine a BACHMANN,
L., E. MULLER, M. L. CARIOU, AND D.
reasonable percentage of the genome.
SPERLICH. 1992. Cloning and characterization of
However, as the insert size increases, there
KM190, a specific satellite DNA family of Drosophila
kitumensis and D. microlabis. Gene 120:267-269.
is an increase in the probability that other
R. J., R. L. HONEYCUTT, AND R. A. VAN DEN
repetitive elements will obscure the seg- BAKER,
BUSSCHE. 1991a. Examination of monophyly of
ments of DNA that can resolve taxonomic
bats: Restriction map of the ribosomal DNA cistron.
boundaries at the desired level. We examBull. Am. Mus. Nat. Hist. 206:42-53 .
ined a cosmid library because if insert BAKER, R. J., C. S. HOOD, AND R. L. HONEYCUTT. 1989.
Phylogenetic relationships and classification of the
sizes of 35 kb could resolve higher taxohigher taxonomic levels of the New World bat famnomic levels (family, superfamily, suborily Phyllostomidae. Syst. Zool. 38:228-238.
der, order) this would be valuable infor- BAKER, R. J., AND D. H. KASS. 1994. Comparison of
mation for designing similar studies for
chromosomal distribution of a retroposon (LINE)
and a retrovirus-like element mys in Peromyscus
isolating higher level taxon-specific semaniculatus and P. leucopus. Chrom. Res. 2:185-189.
quences. Even with 35-kb insert sizes this
R. J., M. J. NOVACEK, AND N. B. SIMMONS.
method appears to be effective in identi- BAKER,
1991b. On the monophyly of bats. Syst. Zool. 40:
fying clones of potential taxon-specific
216-231.
markers that can be expected to address BAKER, R. J., AND M. B. QUMSIYEH. 1988. Methods in
chiropteran mitotic chromosomal studies. Pages
issues for which such markers are appli425-435 in Ecological and behavioral methods for
cable.
the study of bats (T. H. Kunz, ed.). Smithsonian InA drawback to this approach is that cosstitution Press, Washington, D.C.
mid technology is not in widespread use BAKER, R. J., AND H. A. WICHMAN. 1990. Retrotransamong those laboratories in which studies
poson mys is concentrated on the sex chromosomes:
Implications for copy number containment. Evoluof systematic relationships are conducted.
tion 44:2083-2088.
In addition, screening several thousand
BECKMAN, J. S., AND J. L. WEBER. 1992. Survey of hucosmid clones by hybridization can be a
man and rat microsatellites. Genomics 12:627-631.
rather labor-intensive and costly endeavor. BURTON, F. H., D. D. LOEB, C. F. VIOLIVA, S. L. MARTIN,
Thus, the approach presented here will not
M. H. EDGELL, AND C. A. HUTCHISON III. 1986.
Conservation throughout Mammalia and extensive
be easily adopted by all laboratories and
protein-encoding capacity of the highly repeated
its applicability may be somewhat limited.
DNA long interspersed sequence one. J. Mol. Biol.
However, clearly cosmid libraries repre187:291-304.
sent a potentially wealthy source of taxon- DEININGER, P. L. 1989. SINEs: Short interspersed repeated DNA elements in higher eukaryotes. Pages
specific markers to be used in appropriate
619-636 in Mobile DNA (D. E. Berg and M. M.
systematic studies.
ACKNOWLEDGMENTS
We thank Lara Wiggins for assistance in making
figures and preparing the revised manuscript and
Nancy Brown for assistance in library construction.
Funding for this study came from a National Science
Foundation grant (BSR-9107143) to R.J.B.
REFERENCES
ADEGOKE, J. A., U. ARNASON, AND B. WIDEGREN. 1993.
Sequence organization and evolution, in all extant
whalebone whales, of a DNA satellite with terminal
chromosome localization. Chromosoma 102:382388.
Howe, eds.). American Society of Microbiologists,
Washington, D.C.
DEININGER, P. L., AND G. R. DANIELS. 1986. The recent
evolution of mammalian repetitive DNA elements.
Trends Genet. 2:76-80.
DEININGER, P. L., AND V. K. SLAGEL. 1988. Recently
amplified Alu family members share a common parental Alu sequence. Mol. Cell. Biol. 8:4566-4569.
DOVER, G. A. 1982. Molecular drive: A cohesive mode
of species evolution. Nature 299:111-117.
DOVER, G. A. 1986. Molecular drive in multigene
families: How biological novelties arise, spread and
are assimilated. Trends Genet. 2:159-165.
DURFY, S. J., AND H. F. WILLARD. 1990. Concerted evo-
lution of primate alpha satellite DNA: Evidence for
an ancestral sequence shared by gorilla and human
588
SYSTEMATIC BIOLOGY
X chromosome alpha satellite. J. Mol. Biol. 216:555566.
GOUY, M v AND W.-H. Li. 1989. Molecular phylogeny
of the kingdoms Animalia, Plantae, and Fungi. Mol.
Biol. Evol. 6:109-122.
HAMILTON, M. J., R. L. HONEYCUTT, AND R. J. BAKER.
VOL. 4 6
meric tandem repeat in the family Falconidae. Genomics 2: 14-24.
LOVE, J., AND P. DEININGER. 1992. Characterization
and phylogenetic significance of a repetitive DNA
sequence from whooping cranes (Grus americana).
Auk 109:73-79.
1990. Intragenomic movement, sequence amplifi- MCMILLAN, J., T. MAHONY, J. E. N. VERNON, AND D. J.
MILLER. 1991. Nucleotide sequencing of highly recation and concerted evolution in satellite DNA in
petitive DNA from seven species in the coral genus
harvest mice, Reithrodontomys: Evidence from in situ
Acropora (Cnidaria: Scleractinia) implies a division
hybridization. Chromosoma 99:321-329.
contrary to morphological criteria. Mar. Biol. 110:
HAMILTON, M. J., G. HONG, AND H. A. WICHMAN.
323-327.
1992. Intragenomic movement and concerted evolution of satellite DNA in Peromyscus: Evidence from MCMILLAN, J., AND D. J. MILLER. 1990. Highly repeated DNA sequences in the scleractinian coral gein situ hybridization. Cytogenet. Cell Genet. 60:40nus Acropora: Evaluation of cloned repeats as taxo44.
nomic probes. Mar. Biol. 104:483-487.
HUTCHISON, C. A., Ill, S. C. HARDIES, D. D. LOEB, W.
R. SHEHEE, AND M. H. EDGELL. 1989. LINEs and
related retroposons: Long interspersed repeated sequences in the eukaryotic genome. Pages 593-617 in
Mobile DNA (D. E. Berg and M. M. Howe, eds.).
American Society of Microbiologists, Washington,
D.C.
JANECEK, L. L., J. L. LONGMIRE, H. A. WICHMAN, AND
R. J. BAKER. 1993. Organization of repetitive elements in the genome of the white-footed mouse,
Peromyscus leucopus. Mamm. Genome 4:374-381.
KOOPMAN, K. F. 1984. A synopsis of the families of
bats, Part IV. Bat Res. News 25:25-27.
KOOPMAN, K. F. 1993. Order Chiroptera. Pages 137247 in Mammal species of the world, 2nd edition
(D. E. Wilson and D. M. Reeder, eds.). Smithsonian
Institution Press, Washington, D.C.
KORENBERG, J. R., AND M. C. RYKOWSKI. 1988. Human
genome organization: Alu, lines and the molecular
structure of metaphase chromosome bands. Cell 53:
391^00.
MOYZIS, R. K., D. C. TORNEY, J. MEYNE, J. M. BUCK-
INGHAM, J. R. WU, C. BURK, K. M. SlROTKIN, AND W.
B. GOAD. 1989. The distribution of interspersed repetitive DNA sequences in the human genome.
Genomics 4:273-289.
PETTIGREW, J. D. 1986. Flying primates? Megabats
have the advanced pathways from eye to midbrain.
Science 231:1304-1306.
PETTIGREW, J. D. 1991a. Wings or brain? Convergent
evolution in the origins of bats. Syst. Zool. 40:199215.
PETTIGREW, J. D. 1991b. A fruitful, wrong hypothesis?
Response to Baker, Novacek, and Simmons. Syst.
Zool. 40:231-239.
PIERSON, E. D. 1986. Molecular systematics of the Microchiroptera: Higher taxonomic relationships and
biogeography. Ph.D. Dissertation, Univ. California,
Berkeley.
SAMBROOK, J., E. F. FRITSCH, AND T. MANIATIS. 1989.
Molecular cloning: A laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press, Planview, New York.
SIMMONS, N. B. 1994. The case for chiropteran monoposon mys was active during evolution of the Perophyly. Am. Mus. Novit. 3103:1-54.
myscus leucopus-maniculatus complex. J. Mol. Evol. SIMMONS, N. B., M. J. NOVACEK, AND R. J. BAKER.
42:44-51.
1991. Approaches, methods, and the future of the
LONGMIRE, J. L., R. E. AMBROSE, N. C. BROWN, T. J.
chiropteran monophyly controversy: A reply to J. D.
CADE, T. MAECHTLE, W S. SEEGAR, F. P. WARD, AND
Pettigrew. Syst. Zool. 40:239-243.
C. M. WHITE. 1991. Use of sex-linked minisatellite SMITH, J. D. 1976. Chiropteran evolution. Spec. Publ.
fragments to investigate genetic differentiation and
Mus. Tex. Tech Univ. 10:46-69.
migration of North American populations of the SOUTHERN, E. M. 1975. Detection of specific sequencperegrine falcon (Falco peregrinus). Pages 217-229 in es among DNA fragments separated by gel electroDNA fingerprinting: Approaches and applications
phoresis. J. Mol. Biol. 98:503-517.
(T. Burke, G. Dolf, A. Jeffreys, and R. Wolff, eds.). STALLINGS, R. L., A. F. FORD, D. NELSON, D. C. TORNEY,
Birkhauser, Basel, Switzerland.
C. E. HILDEBRAND, AND R. K. MOYZIS. 1991. EvoLONGMIRE, J. L., N. C. BROWN, L. J. MEINCKE, M. L.
lution and distribution of (GT)n repetitive sequences
CAMPBELL, K. L. ALBRIGHT, J. J. FAWCETT, E. W.
in mammalian genomes. Genomics 10:807-815.
LEE, R. N., J. C. JASKULA, R. A. VAN DEN BUSSCHE, R.
J. BAKER, AND H. A. WICHMAN. 1996. Retrotrans-
CAMPBELL, R. K. MOYZIS, C. E. HILDEBRAND, G. A.
EVANS, AND L. L. DEAVEN. 1993. Construction and
characterization of partial digest DNA libraries
made from flow-sorted human chromosome 16.
Genet. Anal. Tech. Appl. 10(3-4):69-76.
LONGMIRE, J. L., A. K. LEWIS, N. C. BROWN, J. M. BUCKINGHAM, L. C. CLARK, M. D. JONES, L. J. MEINCKE,
J. MEYNE, R. L. RATLEFF, F. A. RAY, R. P. WAGNER,
AND R. K. MOYZIS. 1988. Isolation and molecular
characterization of a highly polymorphic centro-
TAUTZ, D., AND M. RENZ. 1984. Simple sequences are
ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res. 12:4127-4138.
VAN DEN BUSSCHE, R. A. 1991. Phylogenetic analysis
of restriction site variation in the ribosomal DNA
complex of New World leaf-nosed bat genera. Syst.
Zool. 40:420^32.
VAN DEN BUSSCHE, R. A., R. J. BAKER, H. A. WICH-
MAN, AND M. J. HAMILTON. 1993. Molecular phy-
logenetics of Stenodermatini bat genera: Congru-
1997
BAKER ET AL.—SOLATION OF DNA TAXON MARKERS
589
Para, Zanderij; Vampyrodes (TK 25083)—Trinidad: St.
George Co., Simla Research Station of the New York
Zoological Society, 6.4 km N Arima; Vampyressa (TK
VAN DEN BUSSCHE, R. A., J. L. LONGMIRE, AND R. J.
BAKER. 1995. How bats achieve a small C-value: 7847)—Nicaragua: Zelaya, 3 km NW Rama; Uroderma
Frequency of repetitive DNA in Macrotus. Mamm. (TK 25256)—Trinidad: St. George Co., Simla Research
Station of the New York Zoological Society, 6.4 km N
Genome 6:521-525.
VAN VALEN, L. 1979. The evolution of bats. Evol. The- Arima. Family Noctilionidae: Noctilio (TK 19077)—
Venezuela: Bolivar, 2.5 km E El Manteco. Family Morory 4:104-121.
moopidae: Pteronotus (TK 27704)—Jamaica: St. Ann's
VASSART, G., M. GEORGES, R. MONSIEUR, H. BROCAS,
Parish, 2 km SW Priory, Circle B Plantation. Family
A. S. LEQUARRE, AND D. CRISTOPHIE. 1987. A sequence in Ml 3 phage detects hypervariable mini- Natalidae: Natalus (TK 15658)—Dominica: St. John
satellites in human and animal DNA. Science 235: Parish, 0.8 km N Toucari. Family Furipteridae: Furipterus (TK 17149)—Suriname: Saramacca, Voltzberg.
683-684.
Family Emballonuridae: Saccopteryx (TK 19493)—
WICHMAN, H. A., S. S. POTTER, AND D. S. PINE. 1985.
Mys, a family of mammalian transposable elements Venezuela: Barinas, 38 km SE Barinas. Family Molosisolated by phylogenetic screening. Nature 317:77- sidae: Tadarida (TK 21984)—Texas: Comal Co., 22.4 km
W New Braunfels, Bracken Cave. Family Rhinolophi81.
dae: Rhinolophus (TK 20037)—New Guinea: East New
Received 28 March 1996; accepted 18 November 1996
Britain Prov., 2 km S Gunanur. Family HipposideriAssociate Editor: Allan Larson
dae: Hipposideros (TK 33178)—Kenya: Coastal Prov.,
Kwale District, Shimba Hills National Reserve, Mwele
Forest, 12 km S, 11 km W Kwale. Family MegaderAPPENDIX
matidae: Megaderma (TK 21288)—Thailand: Uthi
SPECIMENS EXAMINED
Thani Prov., Lansak Dist., Huai Kha Khang Wildlife
Collection (and deposition) locations are given for Sanctuary, Tan Khe Nok, 3.6 km N, 2.6 km W sancindividuals from which genomic DNA was isolated tuary headquarters. Family Nycteridae: Nycteris (TK
(TK = Texas Tech; NK = University of New Mexico). 21529)—Gabon: Estuarie Prov., 2 km SE Cape Esterias.
Order Chiroptera, suborder Microchiroptera, family Family Vespertilionidae: Myotis (TK 19431)—VenePhyllostomidae: Macrotus waterhousii (TK 32184), M. zuela: Barinas, 8 km by road SW St. Barbara. Suborcalifornicus (TK 28962)—Arizona: Pinal Co., Picacho der Megachiroptera, family Pteropodidae: Pteropus
Peak; Desmodus (TK 15376)—Mexico: Yucatan, 1 km N (TK 21493)—Thailand: Chonburi Prov., Panatnikom
Merida; Diaemus (NK 12303), Micronycteris (TK Dist., Wat Loung Temple; Rousettus (TK 27199)—Ke25041)—Trinidad: St. George Co., Simla Research Sta- nya, Western Prov., Kakamega Dist., 6 km S, 6 km W
tion of the New York Zoological Society, 6.4 km N Kakamega; Macroglossus (TK 20239)—Papua New
Arima; Vampyrum (TK 10468)—Suriname: Broko- Guinea: Central Prov., Lakoke Quarantine Stn., 9 km
pondo, Rudi, Kappelvliegueld; Chrotopterus (TK NE Port Moresby; Megaloglossus (TK 21566)—Gabon:
SE Cape Esterias; Nyctimene (TK
17995)—Suriname: Marowijne, Oelamarie; Phyllosto- Estarias, 2 kmNew
Guinea: East New Britain Prov.,
mus (TK 10211)—Suriname: Saramacca, Raleigh Falls; 20095)—Papua
Gela Gela Plantation. Order Dermoptera: CynocephalGlossophaga (TK 20566)—Mexico: Chiapas, 13.1 km SE, us (TK 21407)—Thailand: Surat Thani Prov., Tha
4.0 km E Tonala, Rio Ocuiplapa; Anoura (TK 19335)— Chang Dist., 15 km N, 23 km W Ban Maruan. Order
Venezuela: Barinas, 25 km NW Barinitas; Choeroniscus Insectivora: Crocidura (TK 21587)—Gabon, Estuaire
(TK 10239)—Suriame: Nickerie, Grassalco; Brachyphyl- Prov, 1 km SE Cap Esterias; Scalopus aquaticus (TK
la (TK 15643)— Dominica: St. Paul Parish; Sturnira 29735)—Texas, Montague Co., 4.8 km N, 8 km E Bow(TK 22651)—Peru: Huanuco, 1 km S Tingo Maria; ie. Order Primates: Homo sapiens (TK 30732)—Placenta
Rhinophylla (TK 17728)—Suriname: Saramacca, Tafel- donation from St. Mary's Hospital, Lubbock Co., Lubberg; Centurio (TK 13537)—Mexico: Yucatan, 1 km N bock, Texas; Lemur (TK 26899)—Texas: Tarrant Co.,
Merida; Stenoderma (TK 28361)—Puerto Rico; Artibeus Fort Worth Zoo. Order Rodentia: Mus (TK 28836)—
(TK 32047)—Cuba: Guantamano Prov., Guantanamo Texas: Lubbock Co., 4.8 km W Lubbock.
Bay Naval base; Chiroderma (TK 17371)—Suriname:
ence of data from nuclear and mitochondrial DNA.
Mol. Biol. Evol. 10:944-959.