Post on 28-Mar-2017
Chromosomal evolution in the Drosophila cardini group (Diptera:Drosophilidae): photomaps and inversion analysis
Juliana Cordeiro • Daniela Cristina De Toni •
Gisele de Souza da Silva • Vera Lucia da Silva Valente
Received: 25 November 2013 / Accepted: 2 September 2014 / Published online: 16 September 2014
� Springer International Publishing Switzerland 2014
Abstract Detailed chromosome photomaps are the first
step to develop further chromosomal analysis to study the
evolution of the genetic architecture in any set of species,
considering that chromosomal rearrangements, such as
inversions, are common features of genome evolution. In
this report, we analyzed inversion polymorphisms in 25
different populations belonging to six neotropical species
in the cardini group: Drosophila cardini, D. cardinoides,
D. neocardini, D. neomorpha, D. parthenogenetica and D.
polymorpha. Furthermore, we present the first reference
photomaps for the Neotropical D. cardini and D. parthe-
nogenetica and improved photomaps for D. cardinoides, D.
neocardini and D. polymorpha. We found 19 new inver-
sions for these species. An exhaustive pairwise comparison
of the polytene chromosomes was conducted for the six
species in order to understand evolutionary patterns of their
chromosomes.
Keywords Reference photomap � Heterozygous
inversions � Drosophila parthenogenetica � Drosophila
cardini
Introduction
The comparative analysis of the sequence scaffolds with
the reference photomaps of polytene chromosomes (Scha-
effer et al. 2008) and the currently available 22 genomes
(http://flybase.org) have given rise to an exciting era in the
evolutionary studies of the genus Drosophila (Bhutkar
et al. 2008). Phylogenetic relationships of a number of well
known species groups have been inferred from triads of
overlapping inversions including D. pseudoobscura (Do-
bzhansky and Sturtevant 1938), the virilis group (Throck-
morton 1982), the melanogaster group (Lemeunier and
Ashburner 1976), the repleta group (Wasserman 1992), the
Hawaiian drosophilids (Kaneshiro et al. 1995), and the
willistoni subgroup (Rohde et al. 2006). Although some
studies use only molecular data to infer chromosomal
inversions and phylogenies (Guillen and Ruiz 2012; Pa-
paceit et al. 2013; and many others), some of them still uses
chromosomal maps to help understand the chromosomal
breakpoints composition, transposable elements position,
among other issues. Therefore, chromosomal photomaps
are good tools for phylogenetic studies, and understanding
the origins and maintenance of inversion polymorphisms
will help to reveal mechanisms of genome evolution
(Gonzalez et al. 2007; Brianti et al. 2013; Calvete et al.
2012).
Juliana Cordeiro and Daniela Cristina De Toni contributed data and
results equally to this study.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10709-014-9791-4) contains supplementarymaterial, which is available to authorized users.
J. Cordeiro (&)
Departamento de Ecologia, Zoologia e Genetica, Instituto de
Biologia, Universidade Federal de Pelotas, Pelotas,
Rio Grande do Sul CEP 96001-970, Brazil
e-mail: jlncdr@gmail.com; juliana.cordeiro@ufpel.edu.br
D. C. De Toni
Departamento de Biologia Celular, Embriologia e Genetica,
Universidade Federal de Santa Catarina, Florianopolis,
Santa Catarina CEP 88040-900, Brazil
G. de Souza da Silva � V. L. da Silva Valente
Departamento de Genetica, Instituto de Biociencias,
Universidade Federal do Rio Grande do Sul, Porto Alegre,
Rio Grande do Sul CEP 91501-970, Brazil
123
Genetica (2014) 142:461–472
DOI 10.1007/s10709-014-9791-4
Although most of the studies in the genus Drosophila
are concentrated in the subgenus Sophophora, the subgenus
Drosophila has a rich evolutionary history. The immigrans-
Hirtodrosophila radiation originated in the Paleotropics,
where it initially diversified and expanded, sending the
tripuntata radiation to the Neotropics, where it underwent a
major diversification leading to various species groups.
Besides, the tripunctata and cardini group were among the
many species groups included in this radiation, along with
calloptera. guaramunu, guarani, macroptera, pallidipenis.
rubifrons and sticta (Hatadani et al. 2009; Robe et al.
2010).
Species from the cardini group have been studied since
the 1940s, including research on their taxonomy and dis-
tribution (Sturtevant 1942; Dobzhansky and Pavan 1943;
Stalker 1953; Heed and Russell 1971), crossing patterns
(Heed 1962, 1963), abdominal color polymorphisms (Da
Cunha et al. 1953; Heed and Krishnamurthy 1959; Holl-
ocher et al. 2000a, b; Brisson et al. 2005), chromosomal
inversions, and evolutionary relationships (Martinez and
Cordeiro 1970; Heed and Russell 1971; Brisson et al. 2006;
Cenzi de Re et al. 2010). Moreover, recent collections from
the neotropical Atlantic Forest have shown that these
species have a very significant presence in the Drosophil-
idae community, with the D. cardini, D. cardinoides, D.
neocardini and D. polymorpha being ubiquitous in the
collections (Vilela et al. 2002; Medeiros and Klaczko 2004;
Tidon 2006; De Toni et al. 2007; Roque et al. 2013). This
group consists of 16 described species that inhabit different
areas of Neotropical America (Heed and Russell 1971) and
is characterized by high polymorphic body pigmentation
(Heed and Krishnamurthy 1959; Cordeiro et al. 2009).
According to Bachli (2013), this group is formed by seven
species belonging to the dunni subgroup, whose distribu-
tion is restricted to the Caribbean islands, and nine species
belonging to the cardini subgroup that is distributed from
the southern United States to southern Brazil. The taxo-
nomic studies of the cardini group have documented sev-
eral difficulties in establishing criteria for identifying
species within the group using only morphological char-
acters (Stalker 1953; Heed and Wheeler 1957; Vilela et al.
2002). Moreover, the limits among the groups belonging to
the tripunctata radiation are not always clear (Throck-
morton 1975; Grimaldi 1990; Yotoko et al. 2003; Hatadani
et al. 2009; Robe et al. 2005, 2010).
Phylogenetic relationships within the cardini group have
been proposed using male genital morphology and inter-
crossing tests (Futch 1962; Heed 1962), chromosomal
inversions (Heed and Russell 1971), allozyme polymor-
phisms (Napp and Cordeiro 1981), and nuclear and mito-
chondrial DNA sequence variation (Brisson et al. 2006;
Cenzi de Re et al. 2010). There are general patterns com-
mon to all these inferred phylogenies: D. polymorpha and
D. neomorpha are sister species; D. cardinoides, D. par-
thenogenetica and D. procardinoides form a monophyletic
group; and the D. dunni subgroup of island species also
form a monophyletic group. However, some incongruence
is present, especially with regard to the position of D.
neocardini and the monophyly of the D. cardini subgroup
(Fig. 1). The cardini subgroup is paraphyletic and diverged
from the dunni subgroup 6.6 million years ago (Brisson
et al. 2006). In the non-genetic phylogenies, D. neocardini
is placed together with D. polymorpha and D. neomorpha
(Heed 1962) or alone nearest to the island species (Heed
and Russell 1971). In the consensus genetic phylogenies
(Brisson et al. 2006; Cenzi de Re et al. 2010), D. neocar-
dini is closest to D. cardinoides and D. parthenogenetica.
However, nuclear and mitochondrial data resulted in dif-
ferent topologies (see Cenzi de Re et al. 2010). Considering
patterns of interspecific crossability (Heed and Russell
1971), it is possible that incomplete lineage sorting or
introgression may have caused bias in previous species tree
estimates. Heed and Russell (1971) also performed the
most complete analysis of phylogeny and population
structure of the cardini group and showed that the conti-
nental species from the cardini subgroup were more
diverged genetically from each other than the island spe-
cies were among themselves. Part of the reason for this is
because the cardini group species possess more fixed
inversions. In this way, D. cardini is the most divergent
species of the group, and D. neocardini is closest to the
island species of the dunni subgroup. Nonetheless, the
inversion breakpoints were not shown in their study,
making difficult any comparisons with chromosomes of
other populations. Here, we present and describe new and
improved photomaps and new inversions from the cardini
subgroup species. In addition, we describe a pairwise
comparison analysis of the polytene chromosome rear-
rangements in six of the nine species from the cardini
subgroup: D. cardini, D. cardinoides, D. neocardini, D.
neomorpha, D. parthenogenetica and D. polymorpha.
Materials and methods
Fly stocks
We analyzed different populations from six species: D.
cardini, D. cardinoides, D. neocardini, D. neomorpha, D.
parthenogenetica, and D. polymorpha. All strains were
maintained in the laboratory and reared on standard Dro-
sophila cornmeal culture medium (Marques et al. 1966) in
a controlled chamber (17 ± 1 �C, 60 % rh). The strains
were established using a single fertile female collected
from nature. To ensure correct identification of these spe-
cies which were kindly sent by our collectors,
462 Genetica (2014) 142:461–472
123
collaborators, and the Drosophila Species Stock Center
(University of California, San Diego, USA), we reanalyzed
each stock using the available literature on external mor-
phology and genital characters. A complete list with the
isoline information is given in Online Resource 1, and a
distribution map is given in Online Resource 2.
Cytological preparations
Brain ganglia metaphase chromosomes of all species were
prepared according to Santos-Colares et al. (2002). Sali-
vary glands from third instar female larvae were prepared
according to Ashburner (1989) and three to five female
larvae were analyzed for each isofemale strain.
Polytene chromosome reference photomaps
Slides were examined using a photomicroscope (Zeiss�,
Germany) with phase contrast at 1,0009g magnification,
and images were taken on Kodak ASA 100 color print film.
After comparing the banding pattern of each chromosome
within each species strain, we chose one homokaryotypic
strain each of D. cardini (isostrain CiiSC0003, maintained
in our laboratory) from the Serra do Cipo, Brazil, and D.
parthenogenetica (Stock Center no. 15181-2221.00) from
Sinaloa, Mexico, to produce the first polytene reference
photomaps for these species. Identification of the chro-
mosomes of these species was accomplished by comparing
the banding pattern for each polytene chromosome with
those of the species that already have available reference
photomaps (D. cardinoides and D. polymorpha, Rohde and
Valente 1996; D. neocardini and D. neomorpha, De Toni
et al. 2001a, 2006). To improve the photomaps for D.
cardinoides, D. neocardini and D polymorpha, we used
different photomicrographs from different strains that were
representative of the banding patterns for each polytene
chromosome and followed the banding patterns of previ-
ously published photomaps for each species (Rohde and
Valente 1996; De Toni et al. 2001a). In pairwise analyses
of the polytene chromosomes, we used the photomaps
produced and improved here, as well as the D. neomorpha
photomap (De Toni et al. 2006). The best images were
captured using a film scanner and processed with Adobe�
Photoshop� (v. 10.0.1, Adobe Systems Inc., San Jose,
Fig. 1 Species relationships of
the cardini group. a Species
relationships based on
chromosomal inversions
(modified from Heed and
Russell 1971). b Species
relationships based on edeagus
morphology (modified from
Heed 1962). c Phylogeny based
on gene sequences (modified
from Brisson et al. 2006)
Genetica (2014) 142:461–472 463
123
California, USA). The pairwise chromosome analysis was
conducted by eye using the Photoshop� program. Con-
sidering that D. cardini has 2n = 12 chromosomes while
the other species have 2n = 8 chromosomes, the chromo-
some correspondence was written as the D. cardini chro-
mosome, dash, the chromosome of the other species (e.g.,
chr2-2L).
Results
Metaphase chromosome characterization
Figure 2 depicts the metaphase chromosomes of D. cardini
and D. parthenogenetica. The former species has 2n = 12
chromosomes, and all the chromosomes are acrocentric;
the latter species has 2n = 8 chromosomes with nearly
metacentric autosomes and an acrocentric X chromosome,
as observed for the other species in the group (Heed and
Russell 1971). The recognition of the XY pair in the
metaphase preparations was done by analyzing two char-
acteristics: the heteropicnotic state of the Y chromosome
and the tendency of the homologous chromosomes to be
next to each other. Only D. parthenogenetica showed a
visible secondary constriction in the X chromosomal pair
(Fig. 2d), which was also observed by Stalker (1953). It is
known that this kind of constriction is associated with
rRNA synthesis and can be used as a structural chromo-
somal marker to differentiate closely related species.
Inversion polymorphisms
The heterozygous inversions analyzed in this study are
shown in Figs. 2 and 3. Here, we show the first inversion
detected for D. cardini (Fig. 2e), two new inversions each
for D. neocardini (Fig. 2f, g) and D. cardinoides (Fig. 2h,
i), the two first inversions detected for D. parthenogenetica
(Fig. 2j, k) and 12 new inversions for D. polymorpha
(Fig. 3). The inversions found for each species population
are listed in the Online Resource 1. Considering all
Fig. 2 Metaphase chromosomes from a Drosophila cardini male
(a) and female (b) and from a D. parthenogenetica male (c) and
female (d). Secondary constrictions are indicated by arrows in D.
parthenogenetica chrX. New chromosomal rearrangements for D.
cardini (e), D. neocardini (f, g), D. cardinoides (h, i), and D.
parthenogenetica (j, k). Bar = 10 lm
464 Genetica (2014) 142:461–472
123
inversions identified in this and previous studies (Rohde
and Valente 1996; De Toni et al. 2001a, b), chr3L seems to
be the most polymorphic for heterozygous inversions, both
for D. cardinoides and D. neocardini, with six and two
inversions, respectively. In D. polymorpha, however,
chr3R was the most polymorphic with five inversions.
Reference photomaps of polytene chromosomes
The assembly of the photomaps followed a rigorous and
detailed comparison of each chromosome within each
species strain, making it possible to identify the exact
chromosomal banding pattern of each chromosome. We
also conducted an analysis of each chromosome among
species to insure the correct identification of the
chromosomes. To identify precise locations of banding
patterns and inversion break points, the polytene chro-
mosomes of all species was divided into 20 sections each,
always from the tip to the centromere of the chromo-
somes, and subdivided into ‘‘a’’, ‘‘b’’ and ‘‘c’’ subsections
based on chromosomal banding similarities among spe-
cies and based on the division of previous photomaps
(Rohde and Valente 1996; De Toni et al. 2001a). Chr5-3R
was divided into 19 sections, and chr6-4 (the dot chro-
mosome) constituted the 100th section. The reference
photomaps for D. cardini and D. parthenogenetica were
performed and are provided in Online Resource 3; the
improved reference photomaps for D. cardinoides, D.
polymorpha and D. neocardini are given in Online
Resource 4.
Fig. 3 Newly discovered chromosomal rearrangements in D. polymorpha. Bar = 10 lm
Genetica (2014) 142:461–472 465
123
466 Genetica (2014) 142:461–472
123
Pairwise comparisons of the polytene chromosomes
Comparisons of the chromosomes among species were
done using the new and improved photomaps along with all
of the photographs obtained in this study. For D. neomor-
pha, we used a previously published photomap (De Toni
et al. 2006). To minimize errors in the pairwise chromo-
somal analyses due to observational errors (Wasserman
1992), we performed the same pairwise analysis six times
with four different observers (J. Cordeiro, D. C. De Toni,
G. S. da Silva and V. L. S. Valente). Although D. cardini is
the basal species of the cardini group (Heed and Russell
1971; Brisson et al. 2006; Cenzi de Re et al. 2010) the
banding patterns of the six species studied here generally
resembled those of D. cardinoides. In the pairwise com-
parisons involving D. neomorpha and D. parthenogenetica,
it was difficult to observe similarities. We tried to perform
a chromosomal correspondence with Muller elements by
comparing banding patterns with chromosomes of D.
melanogaster, but these results were ambiguous. It may be
necessary to establish banding pattern homology using
molecular markers for FISH techniques in the future
(Schaeffer et al. 2008; Brianti et al. 2013).
A pairwise analysis of the banding patterns of each
chromosome among these species resulted in 75 compari-
sons (Online Resource 5). The chromosomes used in these
comparisons are shown in Fig. 4, and the break points of
inversions are shown in the chromosomal photomaps
(Online Resource 3 and 4). When comparing the same
chromosome from different species, the tips and base
regions resembled each other. The difficulty in identifying
banding patterns in the medial regions was most likely due
to rearrangements that occurred during the evolutionary
divergence of these species.
Small chromosome regions were detected that could be
used as landmarks. ChrX for all species was recognized by
a puff in the first section and section 20, which remained
attached to the heterochromatic chromocenter (Fig. 4a).
For this chromosome, the precise banding similarities were
generally identifiable, except for the pairwise comparison
between D. cardinoides and D. neocardini. This occurred
for the middle part of chrX in most comparisons. In the
analysis of chrX between D. polymorpha and D. parthe-
nogenetica, we found one region with inverted similarity
where sections from 12b to 14a in D. polymorpha slightly
match with sections from 5a to 8b in D. parthenogenetica
in an inverted direction (Online Resource 5). Moreover, the
only species that has inversions in this chromosome is D.
polymorpha, with the In(X)A/st and In(X)B/st (De Toni
et al. 2001b) and the new In(X)C/st inversions (Fig. 3 and
Online Resource 4).
The first sections of chr2-2L in general shared the same
pattern among species, with strong bands in the tip of each
chromosome followed by a large interband and three strong
bands (Fig. 4b). Moreover, chr2L shared a hexagonal-like
puffed band, used as a landmark that is observed in sec-
tion 23 of D. polymorpha, Sect. 24 of D. cardini and D.
neocardini, and section 25 of D. cardinoides, D. neomor-
pha and D. parthenogenetica. As observed for all chro-
mosomes, the banding pattern of the base of this
chromosome was similar among species, although the
similarity in this region in the comparisons between D.
cardini and D. neocardini, D. cardinoides and D. neocar-
dini, D. neocardini and D. neomorpha, and D. neomorpha
and D. parthenogenetica was difficult to establish (Online
Resource 5). After analyzing photomicrographs from the
base region in different strains of D. cardini and D. neo-
cardini, we could visualize a major similarity of the base
banding pattern of sections 39 and 40 of D. cardini chr2
with sections 59 and 60 of D. neocardini chr2R (Fig. 4
indicated by an asterisk), suggesting the occurrence of a
pericentric inversion between chr2-2L and chr3-2R in the
ancestral lineage of D. neocardini, assuming that D. car-
dini is the basal species of the group. In the search for
inverted gene regions, we found some sequences with
similar banding patterns (Online Resource 5). Neverthe-
less, D. polymorpha is the only species with inversions in
this chromosomal arm, In(2L)A/st and In(2L)B/st, which
are new inversions found in this study (Fig. 3 and Online
Resource 4).
In relation to chr3-2R, no banding regions with relevant
similarities were found that allowed cross identification
among species (Fig. 4c). However, this chromosome fol-
lows the general pattern of similar tips and bases, and D.
polymorpha, D. neocardini and D. cardinoides share great
similarity in their base regions (Online Resource 5). Nev-
ertheless, the comparisons between D. cardini and D.
neocardini, D. cardini and D. neomorpha, and D. neo-
morpha and D. parthenogenetica were difficult, preventing
the establishment of major similarities. Heterozygous
inversions were observed in this chromosome, including
the unique widespread inversion In(3)A/st in D. cardini,
inversion In(2R)A/st in D. parthenogenetica (Fig. 2),
widespread inversion In(2R)A/st (Rohde and Valente
1996) in D. polymorpha, and inversions In(2R)B/st,
In(2R)C/st and In(2R)D/st (De Toni et al. 2001b), in D.
polymorpha (Fig. 3; Online Resource 3 and 4).
Several bands and interbands characterized chr4-3L
with approximately the same width in the first sections
b Fig. 4 Chromosomes of the six cardini subgroup species: a ChrX,
b Chr2-2L, c Chr3-2R, d Chr4-3L, e Chr5-3R, and f Chr6-4. The
bases of the chromosomes are on the left. Dotted lines highlight
information about chrmomosomal similarities that are discussed in
the text. Asterisks in b and c indicate regions that are believed to be
involved in an ancient pericentric inversion (see text)
Genetica (2014) 142:461–472 467
123
(Fig. 4d). The base regions resemble each other among the
species with no evident landmarks. In the pairwise com-
parisons between D. cardini and D. cardinoides, D. poly-
morpha and D. neocardini, and D. neocardini and D.
parthenogenetica, it was difficult to establish similarities in
some parts of the chromosome (Online Resource 5d).
However, regarding the comparisons between D. cardino-
ides and D. neomorpha, D. cardinoides and D.
Fig. 4 continued
468 Genetica (2014) 142:461–472
123
parthenogenetica, and D. neocardini and D. neomorpha,
the whole chromosome seems to be reorganized in a way
that makes the band/interband similarities unidentifiable. In
general, no similarities in the middle of the chromosomes
were detected in these comparisons. Considering all spe-
cies together, this chromosome is the most polymorphic in
the cardini subgroup, with 11 heterozygous inversions.
Drosophila cardinoides has four previously described
inversions, In(3L)A/st, In(3L)B/st, In(3L)C/st, and
In(3L)D/st (Rohde and Valente 1996), and two new
inversions described in our study, In(3L)E/st and In(3L)F/
st. Drosophila neocardini has two inversions, In(3L)A/st
(De Toni et al. 2001a) and the newly described In(3L)B/st,
and D. polymorpha has three newly described inversions,
In(3L)A/st, In(3L)B/st and In(3L)C/st (Figs. 2 and 3;
Online Resource 3 and 4).
As in the other chromosomes, all the tips of the chr5-3R
resembled each other in sections 81–83 (Fig. 4e). In the
comparisons between D. cardini and D. parthenogenetica,
D. neocardini and D. neomorpha, and D. neocardini and D.
parthenogenetica, only the tip region of the chromosomes
showed identified banding similarities (Online Resource
5e). However, in the comparisons between D. cardini and
D. neomorpha and D. polymorpha and D. neomorpha, we
could not identify band/interband similarities along the full
length of the chromosome. In this chromosome, another
new heterozygous inversion for D. parthenogenetica was
observed, In(3R)A/st, as well as a new inversion for D.
neocardini, In(3R)A/st. Drosophila polymorpha has one
previously described inversion, In(3R)A/st (De Toni et al.
2001a), and four new inversions, In(3R)B/st, In(3R)C/st,
In(3R)D/st and In(3R)E/st (Figs. 2 and 3; Online Resource
3 and 4).
Chr6-4 corresponds to the dot chromosomes of all the
cardini group species (Fig. 4f). This chromosome is gen-
erally attached to chrX; however, there were differences in
shape and the number of bands among the species.
Attempts to establish the band number for this
Fig. 4 continued
Genetica (2014) 142:461–472 469
123
chromosome were not always precise; thus, we did not
perform pairwise comparisons among them.
Discussion
The construction of precise photomaps allowing the accu-
rate localization of banding sequences is extremely
important for future studies of gene or transposon mapping
(Cassals et al. 2006; Depra et al. 2009), population struc-
ture (Wallace et al. 2013), and chromosomal or genome
evolution (O’Grady et al. 2001; Ranz et al. 2007; Schaeffer
et al. 2008; Prada et al. 2011; Wallace et al. 2011). Some
studies have shown that polytene chromosome data are
congruent with DNA sequence data when phylogenetic
studies are performed and, when placed in a simultaneous
analysis framework, are shown to be more informative than
nucleotide data (O’Grady et al. 2001; Prada et al. 2011).
Studies of chromosomal evolution of Drosophila species
suggest that the comparison of band/interband pattern is
valuable in evolutionary assays (Heed and Russell 1971;
Lemeunier and Ashburner 1976; Throckmorton 1982;
Wasserman 1992; Kaneshiro et al. 1995; O’Grady et al.
2001; Rohde et al. 2006).
For the genus Drosophila, genome reorganization seems
to be the rule of evolution, although the gene synteny
remains conserved on the same chromosomal arms among
species (Gonzalez et al. 2007; Bhutkar et al. 2008). All
these complex chromosomal conformations suggest rapid
chromosomal evolution (Ruiz and Wasserman 1993;
Guillen and Ruiz 2012). In the cardini group, the occur-
rence of larger chromosomal inversions seems to be more
common than smaller ones. Another source for the
scrambled pattern of the band/interband sequences in these
species may be introgression (Futch 1962; Heed 1962),
reflecting the higher mixture of gene sequences among
these species. As a consequence, phylogenetic incongru-
ence appears to be the norm, in which different markers
appear to support different evolutionary hypotheses (Heed
1962; Heed and Russell 1971; Napp and Cordeiro 1981;
Brisson et al. 2006; Cenzi de Re et al. 2010). It was striking
that we could not identify band/interband similarities in
comparisons between D. polymorpha and D. neomorpha in
whole chromosome comparisons. We expected high simi-
larity in the banding sequences of these two species,
especially regarding the phylogeny of this group when
combining molecular and morphological data (Remsen and
O’Grady 2002; Brisson et al. 2006). Therefore, the com-
bined analysis of the molecular data, morphological data
and polytene chromosomal maps illustrates the importance
of exploring data by both independent and simultaneous
analyses to provide a broader view of the evolutionary
processes acting on this neotropical group of flies.
Drosophila neomorpha and D. parthenogenetica
recently dispersed into South America (De Toni et al.
2005), so their presence in collections is still very low,
despite meticulous efforts of species identification
(Medeiros and Klaczko 2004). In addition, it is difficult
to maintain cardini group species in laboratory condi-
tions, especially these two species (Heed and Russell
1971), and so we had few isostrains for these species in
this study. In our data, D. polymorpha was well repre-
sented, reflecting its ubiquity in nature, followed by D.
neocardini, D. cardinoides and D. cardini. Drosophila
polymorpha is also the most polymorphic species of the
cardini subgroup analyzed here, perhaps indicating that
this species is more of an ecological generalist than the
other species. Heed and Russell (1971) reported that this
species was the second most polymorphic species,
behind D. cardini, which has 26 different paracentric
inversions.
The geographical distribution of the cardini subgroup
encompasses an area extending from Florida, USA to
southern South America. After a meticulous analysis of the
inversion polymorphism of all species in cardini group,
Heed and Russell (1971) proposed that the putative center
of dispersion for this species group was in Central Amer-
ica. These authors found a higher number of polymorphic
inversions than we did; however, the absence of the
breakpoints, drawings, photomaps and photomicrographs
of inversions or chromosomal maps in their study makes
any comparison impracticable. It may be that species
analyzed in our study originated from marginally distrib-
uted populations, which might display a lower degree of
chromosomal polymorphism than those at the center of a
given geographical distribution (Carson 1955; Singh 2008).
This may explain the low chromosomal polymorphism
found in South America populations, for instance, two
inversions for D. cardini, three for D. neocardini, six for D.
cardinoides and 17 for D. polymorpha (Rohde and Valente
1996; De Toni et al. 2001a; and this study). Indeed, D.
neomorpha and D. parthenogenetica were recently identi-
fied in the southern rainforests of Brazil (De Toni et al.
2005), and we found only two inversions for D.
parthenogenetica.
Bhutkar et al. (2008) analyzed the homology of the total
genome sequence of 12 Drosophila species, noting that
95 % of the genetic elements remain in the same chro-
mosome for different species. Genes are rearranged within
each element due to inversions, and different species retain
syntenic blocks of genes, where larger blocks are found in
more closely related species (Schaeffer et al. 2008). This
fact shed some light about which Muller elements would be
analogous of the five arms of the cardini species studied
here, although the in situ hybridization of genes are nec-
essary to confirm this hypothesis. Brianti et al. (2013)
470 Genetica (2014) 142:461–472
123
observed that in D. unipunctata, D. mediopunctata and D.
rhoerae (tripunctata group) there are several conserved
chromomosomal landmarks that can help to easily recog-
nize the Muller B and D elements of these species. These
landmarks are present in chr2/chr2L (Muller B element)
and chr4/chr3L (Muller D element) in cardini subgroup
species. These authors pointed out that in this three tri-
punctata species these two chromosomes are almost ho-
mosequential. The chr3/chr2R is one of the longest in the
cardini subgroup species and also one of the most poly-
morphic. This chromosome would be equivalent to Muller
E element, which is also the largest chromosome among all
species analyzed so far (Brianti et al. 2013). In the cardini
group, phylogenetic relationships based on chromosomal
banding patterns does not correspond to those based on
molecular markers as observed for D. neomorpha and D.
polymorpha. Together with our data, Heed and Russell’s
(1971) results and those of other studies of cardini group
species, we suggest that in situ hybridization techniques are
required to identify Mullers elements, and this will help to
understand the chromosomal evolution of this group
species.
Acknowledgments We would like to thank Dr. William J. Etges
and the reviewers for their valuable comments and help to improve
this manuscript. We would like to thank the fly collectors (Drs. M. F.
D’Avila, H. J. Schmitz, M. S. Gottschalk, C. Rohde, L. Madi-Rav-
azzi, J. Doge, L. Basso da Silva, and W. B. Heed—in memoriam
1926:2007) for their assistance. We also thank Dr. Hope Hollocher for
the D. parthenogenetica strains. This work was funded by grants from
the Brazilian agencies CAPES, CNPq and FAPERGS (10/0028-7).
Conflict of interest The authors declare that they have no conflicts
of interest.
References
Ashburner M (1989) Drosophila: a laboratory manual. Cold Spring
Harbor Laboratory Press, Cambridge, England
Bachli G (2013) Taxodros: the database on taxonomy of Drosophil-
idae. Consulted August 2013. URL: http://www.taxodros.unizh.
ch/
Bhutkar A, Schaeffer SW, Russo SM, Xu M, Smith TF, Gelbart WM
(2008) Chromosomal rearrangement inferred from comparisons
of 12 Drosophila Genomes. Genetics 179:1657–1680
Brianti MT, Ananina G, Klaczko LB (2013) Differential occurrence
of chromosome inversion polymorphisms among Muller’s
elements in the three species of the tripunctata group, including
a species with fast chromosomal evolution. Genome 56(1):17–26
Brisson JA, De Toni DC, Duncan I, Templeton AR (2005) Abdominal
pigmentation variation in Drosophila polymorpha: geographic in
the trait, and underlying phylogeography. Evolution 59(5):104–
1059
Brisson JA, Wilder J, Hollocher H (2006) Phylogenetic analysis of the
cardini group of Drosophila with respect to changes in
pigmentation. Evolution 60:1228–1241
Calvete O, Gonzalez J, Betran E, Ruiz A (2012) Segmental
duplication, microinversion, and gene loss associated with a
complex inversion breakpoint region in Drosophila. Mol Biol
Evol 29(7):1875–1889
Carson HL (1955) The genetic characteristics of marginal populations
of Drosophila. Cold Spring Harb Sym 20:276–287
Cassals F, Gonzalez J, Ruiz A (2006) Abundance and chromosomal
distribution of six Drosophila buzzatii transposons BuT1, BuT2,
BuT3, BuT4, BuT5, and BuT6. Chromosoma 115(5):403–412
Cenzi de Re F, Loreto ELS, Robe LJ (2010) Gene and species trees
reveal mitochondrial and nuclear discordance in the Drosophila
cardini group (Diptera: Drosophilidae). Invertebr Biol
129(353–367):18
Cordeiro J, Valente VLS, Schmitz HJ (2009) Spontaneous melanic
mutant found in a Drosophila neocardini natural population.
Drosoph Inf Serv 92:7–10
Da Cunha AB, Brncic D, Salzano FM (1953) A comparative study of
chromosomal polymorphism in certain South American species
of Drosophila. Heredity 2(7):193–202
De Toni DC, Araujo CB, Morales NB, Valente VLS (2001a)
Reference photomap of the salivary gland polytene chromo-
somes of Drosophila neocardini (Streisinger 1946). Drosoph Inf
Serv 84:88–91
De Toni DC, Heredia FO, Valente VLS (2001b) Chromosomal
variability of Drosophila polymorpha populations from Atlantic
Forest remnants of continental and insular environments in the
State of Santa Catarina, Brazil. Caryologia G Citol Citogenet
54(4):329–337
De Toni DC, Brisson JA, Hofmann PRP, Martins M, Hollocher H
(2005) First record of Drosophila parthenogenetica and D.
neomorpha, cardini group, Heed 1962 (Diptera, Drosophilidae),
in Brazil. Drosoph Inf Serv 88:33–38
De Toni DC, Loureiro MA, Hofmann PRP, Valente VLS (2006)
Reference photomap of the salivary gland polytene chromo-
somes of Drosophila neomorpha (Heed and Wheeler, 1957).
Drosoph Inf Serv 89:73–77
De Toni DC, Gottschalk MS, Cordeiro J, Hofmann PRP, Valente VLS
(2007) Study of the Drosophilidae (Diptera) communities on
Atlantic Forest islands of Santa Catarina State, Brazil. Neotrop
Entomol 36(3):356–375
Depra M, Valente VLS, Margis R, Loreto EL (2009) The hobo
transposon and hobo-related elements are expressed as develop-
mental genes in Drosophila. Gene 448(1):57–63
Dobzhansky T, Pavan C (1943) Studies on Brazilian species of
Drosophila. Bol Fac Filos Cienc S Paulo 36(4):7–72
Dobzhansky T, Sturtevant HA (1938) Inversions in the chromosomes
of Drosophila pseudoobscura. Genetics 23:28–64
Futch DG (1962) Hybridization tests within the cardini species group
of the genus Drosophila. Univ Texas Publ 6205:539–554
Gonzalez J, Casals F, Ruiz A (2007) Testing chromosomal phylog-
enies and inversion breakpoint reuse in Drosophila. Genetics
175(1):167–177
Grimaldi DA (1990) A phylogenetic, revised classification of the
genera in the Drosophilidae (Diptera). Bull Am Mus Natl Hist
197:1–139
Guillen Y, Ruiz A (2012) Gene alterations at Drosophila inversion
breakpoints provide prima facie evidence for natural selection as
an explanation for rapid chromosomal evolution. BMC Genom
13:53
Hatadani LM, McInerney JO, Medeiros HF, Junqueira ACM,
Azeredo-Espin AM, Klaczko LB (2009) Molecular phylogeny
of the Drosophila tripunctata and closely related species groups
(Diptera: Drosophilidae). Mol Phyl Evol 51:595–600
Heed WB (1962) Genetic characteristics of island populations. Univ
Texas Publ Stud Genet 6205:173–206
Genetica (2014) 142:461–472 471
123
Heed WB (1963) Density and distribution of Drosophila polymorpha
and its color alleles in South America. Evolution 17:502–518
Heed WB, Krishnamurthy NB (1959) Genetic studies on the cardini
group of Drosophila in the West Indies. Univ Texas Publ Stud
Genet 5914:155–179
Heed WB, Russell JS (1971) Phylogeny and population structure in
island and continental species of the cardini group of Drosophila
studied by inversion analysis. Univ Texas Publ Stud Gen
6(7103):91–130
Heed WB, Wheeler MR (1957) Thirteen new species in the genus
Drosophila from the neotropical region. Univ Texas Publ Stud
Gen 5721:17–38
Hollocher H, Hatcher JL, Dyreson EG (2000a) Evolution of
abdominal pigmentation differences across species in the
Drosophila dunni subgroup. Evolution 54(6):2046–2056
Hollocher H, Hatcher JL, Dyreson EG (2000b) Genetic and devel-
opmental analysis of abdominal pigmentation differences across
species in the Drosophila dunni subgroup. Evolution
54(6):2057–2071
Kaneshiro KY, Gillespie RL, Carson HL (1995) Chromosomes and
male genitalia of Hawaiian Drosophila: tools for interpreting
phylogeny and geography. In: Wagner WL, Funk AK (eds)
Hawaiian biogeography. Evolution on a hot spot Archipelago.
Smithsonian Institution Press, Washington, pp 57–71
Lemeunier F, Ashburner MA (1976) Relationships within the
melanogaster species subgroup of the genus Drosophila (Soph-
ophora). II. Phylogenetic relationships between six species based
upon polytene chromosome banding sequences. Proc Biol Sci
193(1112):275–294
Marques EK, Napp M, Winge H, Cordeiro AR (1966) A cornmeal,
soybean flour, wheat germ medium for Dmsophila. Dros Inf Serv
41:187
Martinez MN, Cordeiro AR (1970) Modifiers of color pattern genes in
Drosophila polymorpha. Genetics 64:573–587
Medeiros HF, Klaczko LB (2004) How many species of Drosophila
(Diptera: Drosophilidae) remain to be described in the forests of
Sao Paulo, Brazil? Species lists of three forest remnants. Biota
Neotrop 4(1):1–12
Napp M, Cordeiro AR (1981) Interspecific relationship in the cardini
group of Drosophila studied by electrophoresis. Rev Bras Genet
4:537–547
O’Grady PM, Baker RH, Durando CM, Etges WJ, DeSalle R (2001)
Polytene chromosomes as indicators of phylogeny in several
species groups of Drosophila. BMC Evol Biol 1:1–6
Papaceit M, Segarra C, Aguade M (2013) Structure and population
genetics of the breakpoints of a polymorphic inversion in
Drosophila subobscura. Evolution 67(1):66–79
Prada CF, Delprat A, Ruiz A (2011) Testing chromosomal phylog-
enies and inversion breakpoint reuse in Drosophila. The
martensis cluster revisited. Chromosom Res 19(2):251–265
Ranz JM, Maurin D, Chan YS, von Grotthuss M, Hillier LW, Roote J,
Ashburner M, Bergman CM (2007) Principles of genome
evolution in the Drosophila melanogaster species group. PLoS
Biol 5(6):1366–1381
Remsen J, O’Grady P (2002) Phylogeny of Drosophilinae (Diptera:
Drosophilidae), with comments on combined analysis and
character support. Mol Phyl Evol 24:249–264
Robe LJ, Valente VLS, Budnik M, Loreto ELS (2005) Molecular
phylogeny of the subgenus Drosophila (Diptera, Drosophilidae)
with an emphasis on Neotropical species and groups: a nuclear
versus mitochondrial gene approach. Mol Phylogenet Evol
3:623–640
Robe LJ, Valente VLS, Loreto ELS (2010) Phylogenetic relationships
and macro-evolutionary patterns within the Drosophila tripunc-
tata ‘‘radiation’’ (Diptera: Drosophilidae). Genetica 138:725–
735
Rohde C, Valente VLS (1996) Cytological maps and chromosomal
polymorphism of Drosophila polymorpha and Drosophila car-
dinoides. Braz J Genet 19:27–32
Rohde C, Garcia ACL, Valiati VH, Valente VLS (2006) Chromo-
somal evolution of sibling species of the Drosophila willistoni
group. I. Chromosomal arm IIR (Muller’s element B). Genetica
126:77–88
Roque F, da Mata R, Tidon R (2013) Temporal and vertical
drosophilid (Insecta: Diptera) assemblage fluctuations in a
Neotropical gallery forest. Biodivers Conserv 3(22):657–672
Ruiz A, Wasserman M (1993) Evolutionary cytogenetics of the
Drosophila buzzatii species complex. Heredity 70(6):582–596
Santos-Colares M, Goni B, Valente VLS (2002) An improved
technique for mitotic and meiotic chromosomes of Neotropical
species of Drosophila. Drosoph Inf Serv 85:133–136
Schaeffer SW, Bhutkar A, McAllister BF, Matsuda M, Matzkin LM
et al (2008) Polytene chromosomal maps of 11 Drosophila
species: the order of genomic scaffolds inferred from genetic and
physical maps. Genetics 179:1601–1655
Singh BN (2008) Chromosome inversions and linkage disequilibrium
in Drosophila. Curr Sci 94(4):459–464
Stalker HD (1953) Taxonomy and hybridization in the cardini group
of Drosophila. Ann Entomol Soc Am 46:343–358
Sturtevant AH (1942) The classification of the genus Drosophila, with
descriptions of nine new species. Univ Texas Publ 4213:5–51
Throckmorton LH (1975) The phylogeny, ecology and geography of
Drosophila. In: King RC (ed) Handbook of genetics. Plenum,
New York, pp 421–469
Throckmorton LH (1982) The virilis species group. In: Ashburner M,
Carson HL, Thompson Jr JN (eds) The genetics and biology of
Drosophila. Academic Press, London, pp 227–296
Tidon R (2006) Relationships between drosophilids (Diptera: Dros-
ophilidae) and the environment in two contrasting tropical
vegetations. Biol J Linnean Soc 87:233–247
Vilela CR, da Silva AFG, Sene FM (2002) Preliminary data on the
geographical distribution of Drosophila species within morpho-
climatic domains of Brazil. III. The cardini group. Rev Bras
Entomol 2(46):139–148
Wallace AG, Detweiler D, Schaeffer SW (2011) Evolutionary history
of the third chromosome gene arrangements of Drosophila
pseudoobscura inferred from inversion breakpoints. Mol Biol
Evol 28(8):2219–2229
Wallace AG, Detweiler D, Schaeffer SW (2013) Molecular popula-
tion genetics of inversion breakpoint regions in Drosophila
pseudoobscura. G3 3(7):1151–1163
Wasserman M (1992) Cytological evolution of the Drosophila repleta
species group. In: Powell JR, Krimbas CB (eds) Inversion
polymorphism in Drosophila. CRC Press Inc, Boca Raton,
Florida, pp 455–541
Yotoko KSC, Medeiros HF, Solferini VN, Klaczko LB (2003) A
molecular study of the systematics of the Drosophila tripunctata
group and the tripunctata radiation. Mol Phyl Evol 28:614–619
472 Genetica (2014) 142:461–472
123