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