Cytogenetic studies in three species of Lutjanus ( Perciformes : Lutjanidae : Lutjaninae ) from the Isla Margarita , Venezuela

In the present study, three species of Lutjaninae, Lutjanus analis, L. griseus and L. synagris, were analyzed by conventional Giemsa staining, C-banding and silver staining, to reveal active Nucleolus Organizer Regions (NORs). Fluorescent in situ hybridization (FISH) was also applied to establish the number and location of the ribosomal gene clusters (18S and 5S rRNA genes). Counts of diploid metaphasic cells revealed a diploid modal chromosome complement composed of 48 acrocentric chromosomes in both L. analis and L. griseus. Two cytotypes were observed in L. synagris: cytotype I, with 2n=48 acrocentric chromosomes, found in 19 specimens, and cytotype II, with 46 acrocentric chromosomes and one large metacentric, found in two specimens. The large metacentric, which possibly originated from a Robertsonian rearrangement, was not found to be sexrelated. In the three species, constitutive heterochromatin is located in the centromeres of all chromosomes. NORs were detected on the short arms of a single chromosome pair, number 24 in L. analis and number 6 in both cytotypes of L. synagris. In L. griseus, a polymorphism of the NORs number was detected, by both Ag-staining and FISH, as females show a maximum of three NORs, and males a maximum of six NORs. In all species, minor ribosomal genes were found located on a single chromosome pair. The obtained data, along with those previously reported for other five Lutjanidae species, show that a general chromosome homogeneity occurs within the family, but that derived karyotypes based on Robertsonian rearrangements as well as multiple and variable NORs sites can also be found.

In spite of their high number and their ecological and economic importance, cytogenetic studies on Lutjanidae are scarce.In fact, among the 105 recognized species of Lutjanidae, barely five species have been karyotyped to date: Lutjanus argentimaculatus (Raghunath & Prasad, 1980), L. kasmira (Choudhury et al., 1979;Ueno & Takai, 2008), L. sanguineus (Rishi, 1973), L. russelli (Ueno & Ojima 1992), and L. quinquelineatus (Ueno & Takai, 2008).For most of them, only the chromosome number and morphology have been reported and there is no data regarding the chromosomal distribution and composition of the constitutive heterochromatin or numbers and locations of the major and minor ribosomal genes, which have proved to be useful markers in the investigation of the phylogenetic relationships among fish species within a family (Sola et al., 2007).
In the present study, three species of Lutjaninae, Lutjanus analis, L. griseus and L. synagris were analyzed by conventional Giemsa staining and C-banding, and by Fluorescent in situ hybridization with 18S rDNA and 5S rDNA, in order to obtain a fine karyotype characterization, and, thus, chromosome markers which can provide useful information concerning relationships within the family.

Materials and Methods
Eight sexually immature (unsexed) specimens of L. analis, seven specimens of L. griseus (3 males, 3 females, 1 unsexed) and 21 specimens of L. synagris (9 males, 10 females, 2 unsexed) were captured with a fishing trap in the locality of Guayacancito, on Margarita Island, Venezuela.Voucher specimens (Table 1) were deposited at the Ichthyology Collection of the Escuela de Ciencias Aplicadas del Mar (ECAM), Universidad de Oriente.
Twenty four hours before chromosome preparations, the fishes were injected intramuscularly with a yeast glucose solution (Lee & Elder, 1980) for mitosis stimulation.Chromosomes were obtained from kidney cells according to Foresti et al. (1993).C-bands were obtained according to the method described by Sumner (1972), modified by testing different time of exposition to barium hydroxide, from 1 to 180 seconds, in order to enhance the contrast of constitutive heterochromatin on chromosomes.For detection of the active Nucleolus Organizer Regions (NORs), slides were stained with silver nitrate using the method of Howell & Black (1980).The 5S and 18S rDNA sites were identified by FISH according to the method of Pinkel et al. (1986).A sequence of 1800 base pairs of the 18S rRNA gene of Oreochromis niloticus (Nile tilapia), cloned in pGEM-T plasmid, was used as a probe to localize sites for 45S rDNA.PCR products containing 5S rDNA repeats from each species were used as probes for the chromosome mapping of 5S rDNA.DNA was extracted from muscle (Sambrook & Russel, 2001) and the 5S rDNA repeats were generated by Polymerase Chain Reaction (PCR) with the primers 5SA (5'TAC GCC CGA TCT CGT CCG ATC3') and 5SB (5'CAG GCT GGT ATG GCC GTA AGC3') according to Martins & Galetti (1999).
The 18S rDNA and 5S rDNA probes were labeled by nick translation with biotin-14-dATP, following the manufacturer's (Bionick TM Labelling System-Gibco.BRL) instructions.Signals were detected and amplified by a three-round application of Avidin-FITC/biotinilated Anti-avidin.Chromosomes were counter-stained with Propidium Iodide (50µg/ml) diluted in Antifade.
The mitotic figures were photographed using a Motic B400 microscope equipped with a Moticam 5000C digital camera.The fundamental number (NF) of arms was determined considering acrocentrics (A) as having one chromosome arm and metacentrics (M) as having two chromosome arms.FISH metaphases were photographed with a Olympus BX61 photomicroscope equipped with a DP70 digital camera.

Results
The counts of diploid metaphasic cells (Table 1) revealed a modal chromosome complement composed of 2n=48 acrocentric chromosomes (NF=48) in both L. analis and L. griseus and in 19 out of the 21 examined specimens of L. synagris (cytotype I).The two remaining specimens, one male and one unsexed, of L. synagris show a modal count of 2n=47 (NF=48), made up of one large metacentric and 46 acrocentric chromosomes.This karyomorph was named cytotype II.The karyotypes obtained by arranging the chromosomes in order of decreasing size are shown in Fig. 1.The negligible differences in chromosome sizes make it impossible to identify homologous pairs with any certainty, with the exception of a chromosome pair, classified as number 6, in L. analis (Fig. 1a), which shows a secondary constriction when chromosomes are elongated, and chromosome pair number 24 in all the three species, clearly the smallest of the chromosome complements.
In all the three Lutjanus species examined, C-banding (Fig. 2) revealed that the heterochromatin is restricted to the centromeres of all chromosomes, including the large metacentric in cytotype II of L. synagris, though some of them show weaker signals.
The analysis of the nucleolus organizer regions with the Ag-NOR staining technique detected a maximum of two Agpositive paracentromeric signals in L. analis (Fig. 3b) and in both cytotypes of L. synagris (Fig. 3d for cytotype I, data not shown for cytotype II).In L. analis the Ag-signals (Fig. 3b) are located on the secondary constriction, often evident in Giemsa-stained metaphases (Fig. 3a), of chromosome pair number 6.In L. synagris the Ag-signals (Fig. 3d) are located on the smallest chromosome pair number 24 of the complement.In L. griseus, a variable number of paracentromeric Agpositive signals per metaphase was observed, up to two in females (Fig. 4a, b) and up to four in males (Fig. 4c, d).By its morphology and size and for the presence of a secondary constriction (Fig. 4a, b), one of the NOR-bearing chromosome pair in this is likely to be homoeologous to the chromo-    some pair number 6 identified in L. analis (Fig. 3a, b).The remaining signals are located on medium-sized chromosomes.
After FISH with 18S rDNA, in L. analis and in L. synagris two fluorescence hybridization signals were seen at the same chromosome locations detected with Ag-staining, i.e., in paracentromeric positions of chromosome pair number 6 in L. analis (Fig. 5a) and of chromosome pair number 24 in both cytotypes of L. synagris (Fig. 5c, e), confirming that in these species a single pair of NOR-bearing chromosomes is present.In L. griseus (Fig. 6) hybridization with 18S rDNA confirms the NORs sites detected by Ag-staining, but also reveals further positive sites, so that up to three NORs were observed in females (Fig. 6a) and up to six in males (Fig. 6b).All FISH positive chromosomes are medium-sized, thus major ribosomal gene clusters location on chromosome pair 24 can be excluded.
After FISH with 5S rDNA, in all species -L.analis (Fig. 5b), both cytotypes of L. synagris (Fig. 5d, f) and L. griseus (Fig. 6c) -one cluster of 5S rRNA genes was found close to the centromere of a medium-sized acrocentric chromosome pair, which is apparently the same in the three species.
In fish, Robertsonian rearrangements, which, by fusions (or fissions) of two uniarmed chromosomes into one biarmed chromosome (or vice-versa), cause changes in chromosome number, but leave the fundamental number of arms unchanged, are infrequent.However, examples of Robertsonian polymorphism in marine fishes have been reported for species of Gobiidae (Gobius paganellus, Giles et al., 1985;Neogobius eurycephalus, Ene, 2003), Sparidae (Diplodus annularis, Vitturi et al., 1996), Pomacentridae (Chromis insolata and C. flavicauda, Molina & Galetti, 2002), Cottidae (Myoxocephalus scorpius, Yershov, 2005), Gadidae and Pleuronectidae (Gadus morhua and Pleuronectes platessa, Fana & Fox, 1991).In this context, the presence of cytotype II, with a large unpaired metacentric chromosome, found in one male and one unsexed specimen of L. synagris, is quite interesting.In fact, when chromosome differences are restricted to one sex, the presence of sex chromosomes is strongly suggested (Devlin & Nagahama, 2002).As a matter of fact, the presence of 2n=48 acrocentric chromosomes in females and 2n=47 (1 M+46A) in males of L. quinquelineatus was interpreted by Ueno & Takai (2008) as the result of a single Robertsonian fusion which formed a neo-Y chromosome, establishing a multiple sex chro-mosome system of X 1 X 1 X 2 X 2 /X 1 X 2 Y type.In the case reported here for L. synagris, the reduced 2n=47 cytotype II characterizes only two (one male, one immature) of the 21 specimens analyzed, but, among these latter, the remaining eight males present the same 48 acrocentric chromosome cytotype I shown by all ten females and one immature specimens.Thus, the two cytotypes co-exist in different males and the presence of the large unpaired metacentric chromosome does not seem to be associated to sex determination.Due to the frequency of cytotype II, approximately 9.5%, this rearrangement suggests the existence of a chromosomal polymorphism in the studied population of L. synagris.Nevertheless, the possibility that the observed fusion in L. synagris might originate a neo-Y chromosome and that an incipient stage of multiple sex chromosome differentiation, as the one reported in L. quinquelineatus (Ueno & Takai, 2008), has been identified, can not be discarded.
In all the three Lutjanus species examined, the C-positive heterochromatin distribution is restricted to the centromeres  of all chromosomes, a pattern similar to the one reported for the Pacific L. kasmira and L. quinquelineatus (Ueno & Takai, 2008), the only two, among the five karyologically investigated Lutjaninae species, for which data are available.Although the heterochromatin distribution does not identify species-specific chromosome markers, it is worth noting that considerably different exposition times to BaOH, during the C-banding procedure, were applied, from 4 seconds in L. analis, to 30 seconds in L. synagris and up to 180 seconds in L. griseus, a fact which might reflect differences in chromatin condensation levels and/or composition among the species of Lutjaninae here studied.
As far as NORs are concerned, different aspects can be considered.The first one is related to the methodology.In fish, NORs are usually indirectly visualized by silver staining (Nirchio & Oliveira, 2006), which localizes the transcribing major ribosomal genes because silver precipitates in correspondence to a rRNA protein complex synthesized only when NORs are active in the preceding interphase (Hubbel 1985, Sánchez-Pina et al. 1984, Jimenez et al. 1988).However, recent evidence in some fish species (Gromicho et al., 2005) have cast doubt on the accuracy and reliability of Ag in NORs detection, both because other regions, besides NORs, can be stained with Ag or because most of the 28S rDNA sites, as revealed by FISH, were not detected.In the studied species of Lutjaninae, all the Agpositive sites were also FISH-positive, so the technique is NORspecific, but FISH revealed the presence of additional and inactive NORs in only one of them, L. griseus, confirming that this species has a higher NOR variability, both in their number and location, compared to the other congeneric species.As far as this variability is concerned, though a sex-associated trend is identifiable, as males show a higher number of structural NORs, compared to females, a larger sample is needed to verify the extent and the basis of this variability.
A further aspect is related to the cytotaxonomic considerations.A single pair of NOR-bearing chromosomes has been suggested to represent the primitive karyotypical condition in most vertebrate species (Hsu et al., 1975;Schmidt, 1978).In most of the teleost fish displaying the primitive karyotype with 48 acrocentric chromosomes, two terminal NORs near the cen-tromere can be found (Vitturi et al., 1995).Therefore, on one hand, a single NOR bearing chromosome pair, with ribosomal sites interstitially located, as observed in L. analis and L. synagris (present study) and in L. kasmira and L. quinquelineatus (Ueno & Takai, 2008), could be considered a plesiomorphic condition for Lutjanus, whereas the presence of multiple NOR bearing chromosomes, found in L. griseus, would be an apomorphic feature.These data would indicate that L. analis and L. synagris are more closely related, and that the occurrence of duplication and translocation of ribosomal genes in L. griseus might reflect an important mechanism involved in the diversification of this taxon.On the other, when considering NORs location, a different perspective highlights.Indeed, the single NOR-bearing chromosome pair observed in L. analis (chromosome pair 6), as well as one of the NOR-bearing chromosome pairs observed in L. griseus, when pursuing a parsimonious criterion, appears to be homeologous to the one identified both in L. kasmira and L. quinquelineatus (Ueno & Takai, 2008), and, thus, it could be regarded as the primitive condition in the genus.As a consequence, no homeology could be identified with the NOR-bearing chromosome pair of L. synagris.Similar ambiguous results were obtained from molecular and morphological data.Indeed, in a phylogenetic study of Lutjanidae, based on isozyme and morphological data, Chow & Walsh (1992) suggested that L. analis and L. synagris form a clade, which is a sister group of L. griseus.However, subsequent molecular phylogenetic studies, based on mitochondrial DNA (Sarver et al., 1996) have not provided a similar clear-cut subdivision.
FISH experiment with 5S rDNA, revealed their location on a chromosome which is clearly distinct from the one (number 24) bearing major rDNA genes in L. synagris, but, though the difference in size is smaller, also from the NOR bearing chromosome pair number 6 of L. analis and L. griseus.The 5S rRNA gene sites are usually located in separate areas from 45S rRNA genes of the genome in higher eukaryotes (Martins & Galetti, 1999).This is probably because a separate location may permit them to independently evolve, since the divergent functional dynamics of these sequences require physical distancing (Martins & Galetti, 2000).In conclusion, data here obtained for Lutjanus analis, L. griseus and L. synagris, along with those previously reported for L. kasmira and L. quinquelineatus (Ueno & Takai, 2008), show that a general chromosome homogeneity occurs within the family, but that derived karyotypes based on Robertsonian rearrangements as well as multiple and variable NORs sites can also be found.Phylogenetic relationships among species remained unresolved and further data on the other species of Lutjanus are needed to obtain a more general picture of the karyoevolutive trends in the family.

Table 1 .
Number of scored cell and percentages of different diploid counts in specimens of the three species of Lutjanus examined.Undetermined sex (?), male (M), and female (F).