differentiated evolutionary pathways in haemulidae (perciformes): karyotype stasis versus...

RESEARCH PAPER

Differentiated evolutionary pathways in Haemulidae(Perciformes): karyotype stasis versus morphologicaldifferentiation

C. C. Motta Neto • P. A. Lima-Filho •

W. C. Araujo • L. A. C. Bertollo • W. F. Molina

Received: 8 July 2011 / Accepted: 20 September 2011

� Springer Science+Business Media B.V. 2011

Abstract Extensive phenotypic diversity (size, col-

ors and shapes) among species of Haemulidae is

practically dissociated from the conservative cytoge-

netic pattern observed in this family. Detailed analyses

indicate that karyotypic stasis is maintained even under

the scrutiny of different chromosome investigation

methods. Chromosomal banding patterns (endophe-

notype) of five Atlantic species are presented here:

Conodon nobilis, Pomadasys corvinaeformis, Haem-

ulon aurolineatum, H. plumierii and H. steindachneri,

obtained by incorporating the base analog 5-BrdU,

C-banding and staining with base-specific fluoro-

chromes. Despite a few chromosomal specificities,

relative karyotypic conservation was confirmed, cor-

roborating earlier studies on this family. On the other

hand, phenotypic patterns (exophenotype), identified

by geometric morphometrics contrast visibly with the

chromosomal conservation of this group. As such, the

evolutionary rates of chromosomes and body mor-

phology demonstrate clear asynchrony. Possible

causes of karyotypic stasis in Haemulidae are

discussed as well as the sharing of this condition with

other Perciformes.

Keywords Cytogenetics � Geometric

morphometrics � Evolutionary divergence �Chromosome stasis

Introduction

In many groups of marine fish, the speciation process

is not followed by significant karyotype differentia-

tion. This is observed in several Perciformes families

(Molina et al. 2002; Molina 2007), characterized by a

common, extensive and phyletically dispersed karyo-

type composed of 2n = 48 chromosomes predomi-

nantly acrocentric (Klinkhardt et al. 1995; Brum and

Galetti 1997). This karyotypic pattern, considered

basal for this fish order (Galetti et al. 2000), is also

shared by the family Haemulidae (Accioly and Molina

2007; Nirchio et al. 2007). Large panmictic popula-

tions, which would predispose to the maintenance of

intragroup genetic cohesion, or even intrinsic chro-

mosomal characteristics, could be related to karyo-

typic stasis seen in several fish groups (Molina 2007;

Araujo et al. 2010). In many cases, such as in

Haemulidae, this karyotypic stability contrasts with

conspicuous morphological patterns, especially in the

adult phases of development.

A large portion of fish species exhibits rela-

tively small chromosomes, poor in longitudinal bands

C. C. Motta Neto � P. A. Lima-Filho �W. C. Araujo � W. F. Molina (&)

Departamento de Biologia Celular e Genetica, Centro de

Biociencias, Universidade Federal do Rio Grande do

Norte, Natal, RN, Brazil

e-mail: [email protected]

L. A. C. Bertollo

Departamento de Genetica e Evolucao, Universidade

Federal de Sao Carlos, Sao Carlos, SP, Brazil

123

Rev Fish Biol Fisheries

DOI 10.1007/s11160-011-9236-4

and heterochromatic segments. Replication bands,

obtained by incorporating of the base analog

5-Bromo-20-deoxyuridine (5-BrdU) during DNA rep-

lication, may provide resolutive chromosomal char-

acters for identifying cryptic rearrangements, often not

identified by other methodologies (Kasahara 2009).

Indeed, this type of banding has been informative in

the karotypic analysis of a number of groups, such as

Cyprinidae (Zhang and Wu 1985; Hellmer et al.

1991); Salmonidae (Delany and Bloom 1984; Pendas

et al. 1993); Muraenidae (Salvadori et al. 2003),

Scorpaenidae (Giles et al. 1988) and several Charac-

iformes species (Bertollo et al. 1997; Maistro et al.

1999; Daniel-Silva and Almeida-Toledo 2005; Molina

and Galetti 2007), also contributing to phylogenetic

and evolutionary studies (Boron 2003; Sumner 2003).

Morphometric analyses, which enables identifica-

tion of the simultaneous variation of traits related to a

complex body structure (Rohlf and Marcus 1993;

Monteiro and Reis 1999), has contributed to evolu-

tionary biology (Blackith and Reyment 1971), popu-

lation and phylogenetic studies, sex differentiation as

well as intra and interspecific variation (Strauss and

Fuiman 1985; Ehlinger 1991; Vidales et al. 1997;

Fairbairn 1997; Baras 1999; Fulford and Rutherford

2000; Aguirre and Shervette 2005; Rapp Py-Daniel

and Cox Fernandes 2005). The association between

genetic and morphological data has been increasingly

used to understand the processes involved in phylo-

genetic diversification of different groups of organ-

isms (Cheverud 1988; Doebley and Stec 1993; Larson

1998).

The present study investigates chromosomal char-

acters in species of three genera of Haemulidae,

obtained by replicating bands, associated to body

patterns revealed by geometric morphometrics.

Materials and methods

Cytogenetic analyses were carried out with Conodon

nobilis (n = 10, 6 females, 3 males and 1 immature),

Pomadasys corvinaeformis (n = 12, 3 females, 7

females and 2 immatures) and three species of

Haemulon: H. aurolineatum (n = 9, 3 females and 6

males), H. plumierii (n = 8, two females, three males

and three immatures) and H. steindachneri (n = 8,

three females, three males and two immatures),

collected on the coast of Rio Grande do Norte state

(northeastern of Brazil). Samples of C. nobilis,

P. corvinaeformis, H. aurolineatum and H. plumierii

were obtained from two different geographic

areas (5�1301.7300S, 35�9057.8500W and 5�44050.5000S,

35�12010.5200W), while samples of H. steindachneri

were collected in an area further to the south

(5�13011.9700S, 35�2503.8200W).

Specimens were submitted to mitotic stimulation in

vivo with combined antigens (Molina 2001; Molina

et al. 2010), anesthetized with clove oil (Eugenol) and

sacrificed. Mitotic metaphases were obtained from the

anterior portion of the kidney, following the in vivo

preparation method described by Gold et al. (1990).

Heterochromatic chromosomal regions were identi-

fied by C-banding (Sumner 1972) and GC- or AT-rich

segments by CMA3/DAPI fluorochrome staining

(Barros-e-Silva and Guerra 2009). Briefly, slides aged

for 3 days were stained with CMA3 (0.1 mg/ml) for

60 min, restained with DAPI (1 lg/ml) for 30 min,

mounted in glycerol:McIlvaine buffer pH 7.0 (1:1) and

analyzed under epifluorescence microscope equipped

with the appropriate filter set.

Replication bands using the thymine analogue,

5-BrdU were obtained using methodology developed

by Giles et al. (1988). Specimens were submitted to

intraperitoneal inoculation of 5-BrdU (5 mg/ml in

0.9% NaCl solution) at a ratio of 1 ml/100 g of

body weight, 6 h before being sacrificed to obtain

mitotic chromosomes. FPG (Fluorochrome Photolysis

Giemsa) staining was used to reveal RBG (Replication

Bands by Bromodeoxyuridine using Giemsa) bands.

Chromosomal preparations, with base analogue incor-

poration, were stained with Hoescht 33258 solution

(Sigma; 1 mg of Hoescht in 1 ml of methanol and

100 ml of 0.5XSSC) for 40 min in a dark chamber,

washed in distilled water, recovered with a 2XSSC

film and irradiated with ultraviolet light (254 gm) at a

distance of 10 cm, for 1 h, then stained with a 5%

Giemsa solution diluted in phosphate buffer, pH 6.8.

Approximately thirty metaphases were analyzed for

specimen. The best metaphases were photographed

under an Olympus BX50 epifluorescence microscope

equipped with an Olympus DP70 digital image capture

system. The chromosomes acrocentrics were arranged

in the karyotype in descending order of size.

Morphometric analyses were conducted in the fol-

lowing adult specimen samples: C. nobilis (n = 30),

P. corvinaeformis (n = 24), H. aurolineatum (n = 50),

H. plumierii (n = 30) and H. steindachneri (n = 39).


123

The left side of the body was photographed with a Sony

H10 digital camera (8.1 megapixels) mounted on a

tripod at standardized distance and position, eliminating

possible distortions in shape and body size. With respect

to body patterns, eleven common landmarks among

the species were defined. These were located on the

initial (1) and terminal (11) portion of the upper maxilla;

at the anterior (2) and posterior (3) portion at the base of

hard dorsal fin rays; final insertion at the base of the soft

dorsal fin; (4); posterior (5) and anterior (6) base of

the anal fin; insertion of ventral (7) and pectoral fins (8)

and posterior (9) and anterior (10) end of the ocular

cavity.

Procrustes analyses (Rohlf and Slice 1990) were

developed using TPSDig2 software (Rohlf 2006) to

locate landmarks (11 points). Coordinates represent-

ing the spatial position of each landmark were

overlapped using CoordGen software. Analyses of

canonical variables (CV) and MANOVA allowed

morphometric comparisons of the species. Allocation

and cluster tests, using the Jackknife method and

CVAGen6 software, were used to correctly classify

specimens into their respective species. A comparative

deformation matrix of morphological differences

among species was obtained from the mean distribu-

tion of canonic variables through the MorphoJ 1.02b

software (Klingenberg 2011).

Results

The five species analyzed presented 2n = 48 acro-

centric chromosomes (Figs. 1, 2), with symmetric

karyotypes exhibiting small size variation between the

largest and smallest chromosome pairs.

In all species, C-banding showed heterochromatic

blocks, located preferentially in centromeric/pericen-

tromeric regions of all chromosome pairs (Fig. 3a,

partial data) and, occasionally, in other long arm

regions of some pairs of the karyotype. The 18S rDNA

also showed a certain degree of heterochromatic

differentiation (data not shown).

The replication bands obtained exhibited similar

patterns between species, with few differentiated bands

along the chromosomes (Figs. 1, 2), hindering their use

in a more detailed comparative analysis. Based on

previous reports (Motta-Neto et al. 2011a, b), a number

of chromosomes was selected for interspecific compar-

isons, that is, the largest pair of the karyotype (pair 1),

the 5S rDNA bearing pair in all species (pair 5), the pair

bearing an additional 5S rDNA site in C. nobilis (pair

10), the 18S rDNA bearing pair in P. corvinaeformis

(pair 18) and pair 24 in C. nobilis and the three

Haemulon species. These pairs were chosen for their

conspicuous structural or compositional characteristics,

allowing a more effective comparative analysis. Marked

Fig. 1 Karyotypes of

Conodon nobilis (a, b) and

Pomadasys corvinaeformis(c, d), demonstrating the

small number of

differentiated replication

bands along the

chromosomes in different

metaphases of this species.

Bar = 5 lm


123

interspecific conservation was generally observed in

the replication bands on these marker chromosomes,

corroborated by the similar pattern in their C-positive

bands and the co-location of GC-rich regions with 18S

rDNA sites (Fig. 3). The only GC-rich chromosomal

regions (CMA3?/DAPI-) corresponded to 18S rDNA

sites (NORs) present on pair 18 of P. corvinaeformis and

pair 24 of the remaining species.

Cluster analyses of canonical variables indicate that

the variance is distributed mainly by axis 1 (61.51%), 2

(21.39%), 3 (11.41%) and 4 (5.68%). Species belong-

ing to the genus Haemulon were perfectly discrimi-

nated amongst themselves, by both canonical axis 1

and 2 which, taken together, account for 82.90%

of total variance. The greatest morphometric proximity

was sequentially identified between H. plumierii

and both H. steindachneri and H. aurolineatum

(Fig. 4a). Body morphometrics showed signifi-

cant differences (MANOVA axis 1—Lambda =

0.0001, X2 = 1,585.43, df = 72, P \ 0.0001; Axis

2—Lambda = 0.0020, X2 = 998.93, df = 51,

P \ 0.0001), allowing correct discrimination of all

individuals and their respective species, applying the

Jackknife test. Main inter-species differences were

primarily related to head and body height measures.

Thin-plate spline deformation grids for the mean

measures of each species were examined to anatom-

ically describe inter-species shape variations (Fig. 4c).

Fig. 2 Karyotypes of

Haemulon aurolineatum(a, b), H. plumierii (c, d) and

H. steindachneri (e, f),showing the small number

of differentiated replication

bands along the

chromosomes in different

metaphases of these species.

Bar = 5 lm


123

Discussion

The family Haemulidae is characterized by a symmet-

rical and conservative karyotypic pattern (Accioly and

Molina 2007; Nirchio et al. 2007), with little inter-

species chromosome variability. This conservation is

also found in different markers previously analyzed in

this group. In effect, all Haemulidae species analyzed

belonging to the genera Conodon, Pomadasys and

Haemulon exhibit reduced heterochromatic blocks,

located mainly in centromeric/pericentromeric regions

(Nirchio et al. 2007; Motta-Neto et al. 2011a, b),

following the common pattern detected for numerous

Perciformes species (Molina 2007). The same pattern

is repeated in the species investigated here, indicating

their limited applicability in karyotypic differentiation

analysis of Haemulidae. This contrasts with other fish

groups where C-band distribution in chromosomes

may represent of effective population markers (e.g.

Souza et al. 1996). Indeed, this homogeneous pattern in

heterochromatin differs substantially from other fish

groups, where this genome component had an impor-

tant role in karyotypic evolution, as occurs in different

Characiformes species (e.g. Vicari et al. 2008; Cioffi

et al. 2009), revealing polymorphisms and a high

degree of heterochromatin heterogeneity. This evi-

dence corroborates the idea that heterochromatin is not

characterized merely by its degree of condensation, but

also by its organization, molecular composition, asso-

ciated proteins and function (Sumner 2003; Horn and

Peterson 2006).

No base-specific richness was detected in the

studied species, except for nucleolus organizer regions

whose pattern is GC?. In lower invertebrates, ribo-

somal sites, particularly those related to NORs, exhibit

a positive response to GC-specific fluorochromes

(Schmid and Guttenbach 1988), often associated to

C-positive band segments in fish, as occurs in Haemu-

lidae. This occurrence, along with similar chromo-

somal bands obtained by digestion with a number of

restriction endonucleases (Motta-Neto et al. 2011a, b),

suggests marked heterochromatic homogeneity in

these species. A particularly interesting question that

remains unanswered is whether this uniform pattern,

related to repetitive DNA regions, would be associated

to simultaneous chromosomal rearrangements, as

proposed for Pomacentridae species (Molina and

Galetti 2002, 2004), as well as to karyotypic orthose-

lection processes of Perciformes (Molina 2007).

It has been suggested that the systematic occurrence

of certain rearrangements along the chromosomal

evolution by orthoselective processes could lead to

fixation of symmetrical karyotypes, exhibiting simi-

lar-sized chromosomes with the same morphology

Fig. 3 Chromosome pairs 1, 5, 10, 18 and 24 of Conodonnobilis (Cn), Pomadasys corvinaeformis (Pc), Haemulonaurolineatum (Ha), H. plumierii (Hp) and H. steindachneri(Hs), used for inter-species comparative marker analysis.

a C-banding showing the preferential location of C-positive

heterochromatin in the centromeric/pericentromeric region of

the chromosomes; b CMA3 fluorochrome staining depicting

GC-positive regions in pair 18 of P. corvinaeformis and pair 24

of the remaining species; c replication bands showing a similar

pattern among species. Bar = 5 lm


123

(White 1973; King 1981). By contrast, similar kary-

otypes, extensively distributed among species of a

given group, could also indicate the relatively recent

divergence of this group, with insufficient time to

fixate particular chromosomal rearrangements (Sola

et al. 1981). Although this does not explain karyotypic

stasis of several marine families of Perciformes that

emerged in the Tertiary period, this situation cannot be

ruled out for species of Haemulon, whose diversifica-

tion is more recent (Cequea and Perez 1971).

It is well known that several chromosomal banding

techniques are less resolutive in lower than in higher

vertebrates. Thus, replication bands have been used as

a substitute for conventional banding G, Q or R

procedures, which are generally not informative in

fish. In several cases the patterns generated contribute

not only to karyotypic pairing and definition, but also

to sex chromosome analysis, detection of interspecific

homeologies, cytotaxonomy and phylogenetic inves-

tigations (Bertollo et al. 1997; Maistro et al. 1999;

Boron 2003; Daniel-Silva and Almeida-Toledo 2001,

2005; Molina and Galetti 2007; Araujo et al. 2010).

In the Haemulidae analyzed here, where karyotypes

are highly symmetrical with small chromosomes,

replication bands generated a common pattern, reflect-

ing only a slight variation in chromosomal framework

without conspicuous cytotaxonomic markers. How-

ever, in spite of this limitation, data suggest greater

replication band similarity among species of Haemu-

lon, as phylogenetically expected, evidenced by the

relatively higher number of bands.

Nevertheless, individual analysis of some poten-

tially equivalent chromosomes among species, such as

pairs 1, 5, 10, 18 and 24, allowed more effective

interspecific comparison, indicating the maintenance

of large homeologous blocks between them. Despite

the presence of similar structural and replication bands

among species, pair 1 exhibited variable size, around

2 lm in P. corvinaeformis and H. plumierii and 3 lm

in C. nobilis, H. aurolineatum and H. steindachneri,

two or three times larger than the smallest pairs,

respectively. Thus, despite greater karyotypic similar-

ities, some internal reorganization has also taken place

in the chromosomes during the evolutionary process.

Taken together, data obtained by different methodol-

ogies, such as replication banding, base-specific

fluorochrome staining, C-banding and restriction

endonuclease digestion patterns (Nirchio et al. 2007;

Accioly and Molina 2007; Motta-Neto et al. 2011a, b),

suggest that broad structural segments are shared by

Haemulidae genomes. This condition, which has been

indicated for this family, could correspond to a

karyotypic model also valid for other Perciformes

groups. Although information on the maintenance of

phylogenetically syntenic segments shared by Neo-

tropical species is still limited, available data for some

genera (Maistro et al. 1999; Souza et al. 1996, 2001;

Daniel-Silva and Almeida-Toledo 2001, 2005; Vicari

et al. 2008) indicate this may occur.

In contrast to cytogenetic patterns, the morpholog-

ical patterns of Haemulidae species, measured by

geometric morphometrics, demonstrated perfect inter-

species discrimination. The first two canonical vari-

ables explained 82.9% of total variance. Species of

Haemulon were discriminated in relation to both CV

Fig. 4 Geometric morphometrics of analyzed Haemulidae

species. a Species ordering on the first two canonical axes;

b landmarks used in morphometric analyses of the species

(bar = 2 cm); c deformation grid indicating shape variation

between consensus configurations for the species. Vectors show

the deformation direction at each landmark


123

axes. However, it is suggested that the species

analyzed be separated into two groups, one composed

of congeneric species of Haemulon (H. aurolineatum,

H. plumierii and H. steindachneri), and the other, less

discriminated by canonical axis 2, consisting of

representatives of the remaining genera (C. nobilis

and P. corvinaeformis). Morphological data obtained

corroborate the general patterns described for these

species, in which Haemulon is characterized as having

long oblong bodies, while C. nobilis and P. corvinae-

formis have a robust to moderately robust elongated

body (Lindeman and Toxey 2002). Clusters formed

also agree with molecular analyses performed on the

genus Haemulon (Rocha et al. 2008). Graphic repre-

sentation of the data on CV 2 axis, distinguishes

between species with similar feeding habits. Thus, the

group composed of C. nobilis, P. corvinaeformis,

H. plumierii and H. steindachneri, with an imbricated

pattern in relation to CV 2 axis, encompasses species

that feed strictly on macroinvertebrates. The other

more discriminated group consists only of H. auro-

lineatum, which displays more generalist feeding

habits. These are predominantly planktivore, but also

with a macroinvertebrate diet (Lindeman and Toxey

2002). In this case, in addition to reflecting phyloge-

netic patterns, the deformation grid also seems to

indicate functional ecomorphological adaptations,

related to variations primarily in the anterior region

of the body. Indeed, phylogenetic analyses based on

nuclear and mitochondrial genes, associated to the

ecological characteristics of Haemulon species, cor-

roborate historical changes, irrespective of feeding

habits, even among closely related species (Rocha

et al. 2008).

Differentiated evolutionary rates may be attributed

to cytogenetic (endophenotype) and morphological

(exophenotype) divergence in the Haemulidae family,

revealing techytelic evolution (fast-evolving) in terms

of ecomorphological traits. This condition had previ-

ously been determined for Serranidae species (Molina

et al. 2002), whose marked morphological differenti-

ations are present in cytogenetically conservative

species. These data are strongly compatible with the

occurence of ecological speciation in some Haemuli-

dae species (Rocha et al. 2008). In addition to

population isolation, due to strict biogeographic

barriers, evolution in this family may have occurred,

in part, through rapid changes in ecological adapta-

tion after niche fractioning, resulting in particular

morphoecological modifications, without significant

alterations in the karyotypic macrostructure of species.

The karyotypic patterns of Haemulidae presented

here demonstrate extensive chromosomal conserva-

tion, even after different cytogenetic investigation

methodologies, suggesting diversification of shapes

without concomitant fixation of conspicuous chromo-

somal rearrangements. This may also be extensive for

other families of Perciformes, which have also

revealed similar chromosomal patterns to those of

the family Haemulidae.

Acknowledgments The authors thank the Federal University

of Rio Grande do Norte, CNPq (Process No. 556793/2009-9),

and IBAMA (Process No. 19135/1) for supporting this study,

Jose Garcia for taxonomic identification of the specimens and

Pablo Martinez for assistance with geometric morphometrics

analysis.

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