Helgol Mar Res (2012) 66:363–370 DOI 10.1007/s10152-011-0276-x

ORIGINAL ARTICLE

Chromosomal diVerentiation and speciation in sister- of () from the Western Atlantic

Wagner Franco Molina · Gideão Wagner Werneck Felix da Costa · Marcelo de Bello CioY · Luiz Antonio Carlos Bertollo

Received: 31 May 2011 / Revised: 30 August 2011 / Accepted: 8 September 2011 / Published online: 22 September 2011 © Springer-Verlag and AWI 2011

Abstract In the tropical Atlantic, the ichthyofauna diVerentiating status of these species, possibly achieved between the coast of Brazil and the Caribbean regions, under the conditions of allopatry due to the Amazon/Orinoco divided by the Amazon barrier, is very similar presenting barrier, showing chromosomal peculiarities in Grammatidae several geminate species, including , species when compared to other groups of Perciformes. endemic in Brazil, and its Caribbean counterpart Gramma loreto. Morphological and molecular studies have helped Keywords Marine Wsh · Chromosomal markers · establish evolutionary patterns that sister-species of these Chromosomal evolution · Allopatric speciation · two marine habitats are subjected to. However, their chro- Marine barriers mosomal characteristics are only beginning to be better characterized. Accordingly, a comparative cytogenetic analysis was carried out in G. brasiliensis and G. loreto, Introduction seeking evidence of cytotaxonomic markers implicated in the karyotypic diversiWcation of these species and likely The distribution patterns of marine biodiversity are com- associated with speciation events. Heterochromatic regions plex, resulting from vicariance events and dispersion and their aYnity to Xuorochromes GC- or AT-speciWc were potential of species as well as local ecological and adap- identiWed, as well as the distribution of ribosomal DNA tive conditions. In the tropical Atlantic, the recognition of sites in chromosomes, either by silver nitrate impregnation these patterns has been established incrementally (e.g., (Ag-NORs) or dual-color mapping with 18S and 5S Rocha 2003; Floeter et al. 2008), including descriptions rDNA probes. While displaying the same diploid number, and revalidations of species endemic to the Brazilian 2n = 48 chromosomes, considered basal for Perciformes, coast (Rocha and Rosa 2001; Moura and Castro 2002; the two species diVered in karyotype structure, showing Moura and Lindeman 2007). Advances in reef Wsh karyotypic formulas and species-speciWc heterochromatin research have identiWed species previously considered pattern. The cytological characters found support the endemic to Brazil in the Southern Caribbean (Rocha et al. 2002). At the same time, species previously considered the same as to those of the Caribbean have been identiWed as new on the Brazilian coast (e.g., Rocha and Rosa 1999; Communicated by H.-D. Franke. Moura and Lindeman 2007). In many cases, molecular (e.g., Bowen et al. 2001; Rocha et al. 2002; Tornabene W. F. Molina (&) · G. W. W. F. da Costa et al. 2010) and cytogenetic patterns (Nirchio et al. 2005; Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte Rossi et al. 2005; Motta-Neto et al. 2011) have helped (UFRN), Natal, RN, Brazil identify genetic diversity among species and populations e-mail: [email protected] of these marine provinces. In the tropical Atlantic, strong environmental pres- M. de Bello CioY · L. A. C. Bertollo Departamento de Genética e Evolução, sure and extraction occurring in coastal areas of Brazil Universidade Federal de São Carlos, São Carlos, SP, Brazil and the Caribbean have endangered several small 123 364 Helgol Mar Res (2012) 66:363–370 benthic species (Leão and Dominguez 2000; Ferreira et al. 2005; PandolW et al. 2003). Cryptobenthic Wsh stand out for their trophic importance (Depczynski and Bellwood 2003). However, most of these species are not well studied for any biological parameters. Generally, they have a short larval period and some species, even in adulthood, appear to remain near the coast (Beldade et al. 2007). Highlighted here are Wsh from the Gram- matidae family (Ferreira et al. 2005), a monophyletic group comprising 13 species with distribution restricted to the Western Atlantic (Gill and Mooi 1993). In this group, the genus Gramma is composed of only Wve spe- cies (Böhlke and Randall 1963; Starck and Colin 1978; Sazima et al. 1998; Victor and Randall 2010), inhabiting shallow waters, such as G. loreto and G. brasiliensis, or deeper regions such as G. melacara, G. linki and G. dejongi (Victor and Randall 2010). Gramma loreto, a small planktivorous species present in the Caribbean Fig. 1 Schematic presentation of the areas of occurrence of (Böhlke and Randall 1963), and G. brasiliensis, endemic to (a) Gramma loreto and (b) the sister-species Gramma brasiliensis along the Caribbean and Brazilian coast. Highlighted between these the Brazilian Western Atlantic (Sazima et al. 1998), have two areas is the zone of inXuence of discharges from the Orinoco and vibrant coloration patterns, with a distinctive purple and yel- Amazon rivers into the Atlantic Ocean. The areas (1) and (2) corre- low bicolor body (Fig. 1). These species are increasingly spond to Rocas Atoll and Fernando de Noronha Archipelago, respec- exploited by the aquarist market, putting them at risk tively (Monteiro-Neto et al. 2003; Gasparini et al. 2005; Bruckner 2005). Indeed, the harvesting eVect in some areas of north- east Brazil has reduced the population size of G. brasilien- Materials and methods sis, given their low dispersal and recolonizing capacity. In addition, characteristics such as parental care and short lar- Specimens and chromosome preparations val stage may also increase the risk status of this species (Ferreira et al. 2005). Phylogenetically, the position of Five G. loreto specimens (two males, two females, and one Grammatidae among Perciformes remains uncertain. They immature), obtained from ornamental Wsh importers, and are considered to belong to the group with ten specimens of G. brasiliensis (Sazima, Gasparini and demersal eggs (Mooi 1990). Moura 1998) (Wve males, three females, and two imma- Although fauna similarities and endemism between the ture), from the coast of Salvador (12°58ЈS/38°31ЈW), Bahia coast of Brazil and the Caribbean are recognized, chromo- state, Northeast Brazil, were employed in chromosome somal diVerences between species and populations of analysis. these regions have been neglected. In this study, two spe- The specimens were subjected to mitotic stimulation cies of Grammatidae, G. loreto () and using attenuated antigen compounds, for a period of 24 to G. brasiliensis (Brazilian Royal Gramma), were com- 28 h, according to Molina (2001) and Molina et al. (2010). pared cytogenetically with the use of conventional stain- The were then anesthetized with clove oil (euge- ing, C-banding, identiWcation of nucleolar organizer nol) and sacriWced for the removal of anterior kidney frag- regions by silver nitrate (Ag-NORs), staining with Xuoro- ments and sexed by macroscopic and microscopic chrome GC- and AT-speciWc and chromosome mapping examination of the gonads. Chromosome preparations were by dual-color Xuorescence in situ hybridization (dual- obtained from cell suspensions of anterior kidney, through color FISH) with 18S and 5S rDNA probes. The results the in vitro interruption of the mitotic cycle, according to showed that these species constitute a suitable model for the method proposed by Gold et al. (1990). investigating chromosomal rearrangements in marine populations, revealing Grammatidae chromosomal singu- Chromosome banding larities in relation to other Perciformes. Meanwhile, allowed to analyze the relationship between chromosomal Nucleolar organizer regions (NORs) were identiWed using rearrangements and speciation, an issue that is beginning the silver impregnation method (Ag-NORs), as described to be more widely discussed (Molina 2007). by Howell and Black (1980). C-banding according to

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Sumner (1972), with minor modiWcations, was used to Results detect C-positive heterochromatin. Sequential staining with the Xuorochromes chromo- Both species have the same diploid number (2n =48), Ј mycin A3 (CMA3) and 4 ,6-diamidino-2-phenylindole with karyotypes 4 m + 6st + 38a in G. loreto and 6 m + (DAPI) was used to identify GC- and AT-rich chromo- 6st + 36a in G. brasiliensis (Fig. 2a, b). The smallest chro- somal regions, respectively (Barros-Silva and Guerra mosome pair (pair no. 5 in G. loreto and pair no. 6 in W 2009). Slides aged for 3 days were stained with CMA3 G. brasiliensis) is signi cantly shorter than all other karyo- (0.1/mg/ml) for 60 min and restained with DAPI (1 g/ml) type pairs. In this pair, it is diYcult to identify the correct for 30 min. They were then mounted with antifading position of the centromere due to its small size (>1.5 m). Vectashield and kept at 4°C for 5 days until analysis However, it was classiWed as subtelocentric, given data with an Olympus BX50 epiXuorescence microscope, obtained in the majority of preparations examined. Thus, using appropriate excitation Wlters. Joint identiWcation the two species diVer in the number of m/sm chromosomes, X V of positive uorochromes CMA3 and DAPI areas was which gives them di erent karyotypic formulas, as well as obtained from the overlap of sequential images stained diVerent fundamental number (FN). In fact, G. loreto is of the same metaphase, using Adobe Photoshop CS5 characterized by FN = 58 and G. brasiliensis by FN = 60, software. considering m/sm chromosomes, bi-armed st chromo- somes, and one-armed acrocentric chromosomes. Fluorescence in situ hybridization and karyotype analysis Only one pair of Ag-NORs was detected, showing termi- nal location on the short arms of the largest subtelocentric Fluorescence in situ hybridization (FISH) was per- pair (pair no. 3 in G. loreto and pair no. 4 in G. brasiliensis) formed under high stringency conditions on mitotic (Fig. 2a, b, highlighted). Similarly, 18S rRNA genes were chromosome spreads according to Pinkel et al. (1986). mapped in the same position and on the same chromosome The 5S and 18S rDNA sequences were detected by dual- pairs in both species (Fig. 2 g, h). In turn, 5S rRNA genes color FISH analysis. The two ribosomal sequences were were mapped in both species at the terminal extremity of isolated from the Hoplias malabaricus (Teleostei, Char- the chromosomes, located on the long arms of pairs 9 and aciformes) genome. The 5S rDNA repeat copy included 10 of G. loreto and G. brasiliensis, respectively, and the 120 base pairs (bp) of the 5S rRNA encoding gene and short arms of pairs 14 and 15 G. of loreto and G. brasilien- 200 bp of the non-transcribed spacer (NTS) (Martins sis, respectively (Fig. 2 g, h). et al. 2006). The 18S rDNA probe corresponded to a Heterochromatic blocks were highlighted in the centro- 1,400 bp segment of the 18S rRNA gene, obtained via meres of practically all the chromosomes; however, they PCR from nuclear DNA (CioY et al. 2009). The 5S and were more noticeable in G. brasiliensis than in G. loreto + 18S rDNA sequences were cloned into the pGEM-T (Fig. 2c, d). GC-rich heterochromatin distribution (CMA3 / plasmid (Promega, Heidelberg, Germany) and propa- DAPI¡) with NOR sites was clearly characterized in both gated in DH5 E. coli competent cells (Invitrogen, San species. This correspondence was also observed for 5S Diego, CA, USA). The 5S rDNA probe was labeled with rDNA sites, albeit less marked. In turn, most heterochro- biotin-14-dATP by nick translation according to manu- matic regions in other G. loreto and G. brasiliensis chromo- facturer’s recommendations (BioNick™Labeling Sys- somes also exhibited a GC-rich composition, responding tem; Invitrogen, San Diego, CA, USA). The 18S rDNA positively to chromomycin A3 staining (Fig. 2c–f). was labeled by nick translation with DIG-11-dUTP, according to manufacturer’s instructions (Roche, Mann- heim, Germany). Discussion Approximately thirty metaphases were analyzed for each specimen to determine diploid number and karyotype. The Chromosome conservation £ diVerentiation best metaphases were photographed with an Olympus and karyotype evolution BX50 epiXuorescence microscope, equipped with a DP70 Olympus digital image capture system, used to determine Characteristics, such as 2n = 48 acrocentric chromo- karyotypes. somes, unique NORs, reduced heterochromatic content, The chromosomes were grouped into metacentric (m), and restricted to centromeric/terminal regions, are likely subtelocentric (st), and acrocentric (a) according to the basal for Perciformes (Brum and Galetti 1997; Molina position of the centromere (Levan et al. 1964) and arranged 2007). Among the cytogenetically conserved groups in in descending order of size. Considering the diYculty of this order, such as Sciaenidae, some or all of these charac- precise m and sm chromosome identiWcation, they were teristics are present in more than 85% of the species grouped together in the karyotype. analyzed (Accioly and Molina 2008). In this context, 123 366 Helgol Mar Res (2012) 66:363–370

Fig. 2 Karyotype of G. loreto (a, c, e, g) and G. brasiliensis (b, d, f, h). Conventional Giem- sa staining (a, b), highlighting Ag-NORs sites on the short arms of pairs 3 and 4 of G. loreto and G. brasiliensis, respectively; C-banding showing heterochro- matin in centromeric chromo- somal regions (c, d); Sequential staining with Xuorochromes CMA3/DAPI (e, f), showing the composition of most GC-rich heterochromatin blocks and location of ribosomal genes by dual-color FISH with rDNA 18S (pair 3 of G. loreto and pair 4 of G. brasiliensis) and 5S (pairs 9 and 14 of G. loreto and pairs 10 and 15 of G. brasiliensis) (g, h)probes. Bar =5m

sister-species karyotypes of Grammatidae, G. loreto and chromosomes (FN = 60). Indeed, the most diagnosed G. brasiliensis, despite their basal chromosome number cytotaxonomic marker for these species is found among remaining unchanged (2n = 48), contrast in other aspects the meta-submetacentric and acrocentric chromosomes. with the widely present conservatism of Perciformes. While 4 m-sm and 38a occur in G. loreto, 6 m-sm and 36a Whereas G. loreto exhibits a larger number of acrocentric occur in G. brasiliensis, likely as a result of pericentric chromosomes (FN = 58) and, therefore, more related to inversion, currently Wxed in the homozygous condition of the karyotype considered basal for the group, G. brasili- the latter species, changing one pair of acrocentric chro- ensis shows a higher number of two-armed acrocentric mosomes into one pair of m-sm chromosomes. 123 Helgol Mar Res (2012) 66:363–370 367

It seems apparent that pericentric inversions correspond erochromatin dispersed in diVerent chromosomes of the to the main chromosomal rearrangements found in Perci- karyotype in both species, in addition to ribosomal sites, formes (Cano et al. 1982; Ozouf-Costaz et al. 1991; Galetti contrasts with the pattern usually observed in Perciformes, et al. 2000), as well as in other phylogenetically diverse where a neutral response to AT- or GC-speciWc Xuoro- marine groups, such as Tetraodontiformes (Sá-Gabriel and chromes is observed (except the major rDNA sites), indi- Molina 2005; Noleto et al. 2007; Martinez et al. 2010), cating a probably apomorphy of the family Grammatidae. Anguilliformes (Takai and Ojima 1985; Vasconcelos and However, in addition to these diVerentiating characters Molina 2009), and Batrachoidiformes (Brum et al. 2002; between G. loreto and G. brasiliensis, other chromosomal Nirchio et al. 2002; Costa and Molina 2009), indicating markers were conserved in both species, such as the num- their eVective participation in the karyotypic evolution of ber and location of Ag-NORs and in turn 18S rDNA and 5S these groups. rDNA sites. There is a clear association between chromo- More recently, the role of inversions in the evolutionary somes at these sites in the two species, suggesting homeol- process has been reassessed. After inversion occurs, it can ogy between them. Thus, while some of the chromosomal be lost in the polymorphic state or, under the proper condi- characteristics of G. loreto and G. brasiliensis underwent tions, spread in the population until it is Wxed. Even though diVerential processes, others remained stable and conserved few genes related to traits adapted to new environmental/ throughout their evolutionary history. Similar observations climatic conditions and the speciation process were identi- were also made for pairs 5 and 6 of G. loreto and G. brasil- Wed in the inversions, there are growing indications of their iensis, respectively, which can be considered microchromo- decisive role in these events. Inversions maintain areas of somes because of their reduced size (1.5 m), markedly imbalance between alleles in loci within or inXuenced by diVerent from the other pairs of the karyotype. Microchro- these rearrangements, leading to an adaptive condition, pri- mosomes have often been found in diVerent groups of ver- marily along environmental gradients (for a review see tebrates (King 1990), primitive Wsh (Rock et al. 1996), and HoVmann and Rieseberg 2008). This could occur, particu- various marine Perciformes (Takai and Ojima 1987). In larly in relation to possible historical expansion and adapta- both Gramma species, these chromosomes were constant in tion to new environments more toward the south, for all metaphases analyzed, thereby characterizing them as G. brasiliensis, compared to G. loreto. The diVerent occur- regular components of their chromosomal lots and exclud- rence of pericentric inversions among coastal marine spe- ing them as possible B or supranumerary chromosomes, cies and their likely derived insular forms have also been which can vary between specimens or even among the cells identiWed in other marine Perciformes, such as in species of of an individual (Carvalho et al. 2008). This karyotypic the genus Stegastes. In this case, S. sanctipauli and trait is shared by G. loreto and G. brasiliensis, allowing us S. rocasensis, inhabitants of ecologically diverse areas far to infer about the ancestry of their presence, likely prior to from the mainland, had a larger number of detectable peri- the speciation process that diVerentiated these species, centric inversions when compared to land-based forms thereby constituting a phylogenetic marker for these forms. (Molina 2007), demonstrating the relevant role of these rearrangements in the evolutionary history of these groups. Chromosomal diVerentiation and speciation In general, reduced heterochromatic/centromeric/telom- eric content corresponds to the widely dispersed pattern Fish fauna comparisons between Atlantic regions have among Perciformes (Molina 2007). A few exceptions are pointed to the Caribbean as a center of diversity and active known, such as a species of Centropyge (Pomacanthidae) exporter of a range of reef Wsh. Mitochondrial sequence on the coast of Brazil, which exhibits conspicuous het- analyses in Caribbean populations of the pomacentrid Chr- erochromatic blocks on the short arm of a number of omis multilineata support this idea, indicating that they are subtelocentric chromosomes (AVonso and Galetti 2005). older than in other Atlantic regions, including Brazil C-positive heterochromatin in G. loreto and G. brasiliensis (Rocha 2003). Historical environmental variations caused maintains the preferential pericentromeric pattern, but dis- by glacial events (e.g., Gysels et al. 2004), as well as the tributed to practically all chromosomes of the karyotype, in emergence of the Amazon/Orinoco river barrier (Rocha addition to the short arms of NOR-bearing subtelocentric 2003, Rocha et al. 2008), had a potential inXuence on low- chromosomes. However, there is predominance of more vagility cryptobenthic species inhabiting shallow waters, conspicuous heterochromatic blocks in G. brasiliensis, leading to population restrictions and the Wxation of genetic underscoring a diVerent heterochromatinization process divergences, such as the chromosomal modiWcations when compared to G. loreto. In this sense, G. brasiliensis observed in the Gramma species analyzed here. Indeed, the once again stands out as a relatively more diVerentiating role of glacial events in fractioning and restricting popula- karyotype, reinforcing its condition, derived from the genus tions is considered relevant for the chromosomal diversiW- Gramma. On the other hand, the occurrence of GC-rich het- cation and transitory polymorphisms present in marine 123 368 Helgol Mar Res (2012) 66:363–370 species of the genus Chromis (Molina and Galetti 2002). conditions of geographic isolation than in sympatric or Moreover, the barrier formed by the discharge of the Ama- even parapatric conditions. zon/Orinoco Rivers (t10 m.a) has also played a crucial The allopatric eVects on the karyotype of these species role in the evolution of reef Wsh in the tropical Atlantic indicate forms with large steps of divergence and potential (Briggs 1974; Robertson et al. 2006). This barrier is consid- degree of postzygotic reproductive isolation. It is known that ered the primary cause of sister-species formed in the chromosome rearrangements, such as inversions, can repre- diVerent families of reef Wsh (Floeter and Gasparini 2001; sent eVective postzygotic reproductive isolation mechanisms Rocha 2003), promoting an eVective or suYcient impedi- (Noor et al. 2001; Brown et al. 2004). In this sense, repro- ment to gene Xow between the Caribbean and Brazilian ductive behavior analysis of G. loreto and G. brasiliensis coast, mainly in shallow water species. under sympatric conditions in nature would be highly eluci- Nearly all Grammatidae species are found in the Carib- dative, although such a possibility seems to be very remote, bean, suggesting that this region, similarly to what is considering the biogeographic isolation barrier between observed in other groups (Rocha et al. 2008), is also the them and the diYcult transposition for any of these species. diversiWcation center of this family. On the other hand, G. brasiliensis, a species endemic to the Brazilian coast, Final considerations likely represents a more recent derived form than G. loreto. This hypothesis is supported by the more conservative The karyotypic pattern of sister-species G. loreto and karyotypic characteristics of G. loreto, when compared to G. brasiliensis, in addition to expected similarities, also G. brasiliensis. Indeed, cytogenetic data indicate that shows a speciWc chromosomal diagnosis. Data strongly sug- G. brasiliensis exhibits a more derived karyotype, consider- gest that G. brasiliensis is a more recent species, derived ing its karyotypic formula and the more conspicuous from G. loreto through a process of allopatric speciation heterochromatinization of its chromosomes. aVorded by biogeography isolation from the Amazon/Ori- Sympatric and parapatric speciation in marine Wsh have noco barrier. been increasingly reported (Rocha 2003; Rocha and Bowen In this case, chromosomal diVerences between these two 2008), demonstrating that they are more frequent events Gramma species reXect about ten million years of evolu- than were previously believed. One possible example of tionary diVerentiation. Very little is known about the chro- sympatric speciation was reported in Grammatidae. The mosomal alterations involved in the speciation process in a species G. dejongi, inhabiting the deep waters oV Cuba, marine environment. Although prezygotic barriers can be occurs within the distribution area and in sympatry with considered isolation mechanisms between species of Perci- G. loreto. The two species share morphological characteris- formes (Kocher 2004), postzygotic chromosomal barriers tics and have similar mtDNA barcodes (Victor and Randall may have been established between the two Gramma spe- 2010), suggesting a valid species with recent sympatric spe- cies analyzed, hindering or even preventing possible ciation. On the other hand, the areas of inXuence belonging hybridizations and introgression between them. Thus, cyto- to the Amazon/Orinoco Rivers demonstrate marked ecolog- genetic studies in diVerent regions of the Atlantic might ical eVects on G. loreto, and its germinate species, G. bra- provide informative data for diagnosing interspeciWc and/or siliensis, promoting a conspicuous environmental exclusion interpopulation diVerentiation, especially in the investiga- zone between them, as well as between any other Grammat- tion of chromosomal events that may have promoted the idae species. This barrier probably led to reproductive iso- speciation process in this environment. lation between populations of G. loreto for enough time to allow diVerentiation and allopatric speciation between Acknowledgments We are grateful to the National Council for Sci- W W them. The premise of allopatric speciation is more plausible enti c and Technological Development (CNPq) for nancial support (Process No. 556793/2009-9), IBAMA (Process No. 19135/1), Federal for shallow water species and specialized habits, such as University of Rio Grande do Norte for providing the means to conduct G. loreto, where the Amazon/Orinoco barrier is a physical, this study and M. F. Molina for assisting in chromosome preparation in physiological, ecological, and possibly insurmountable the state of Bahia. obstacle, even during glaciations events. Two aspects sup- port this hypothesis. The Wrst is phylogenetic (phylogeo- graphic), evidenced by the absence of Grammatidae species References along the Brazilian coast, as well as in the zone aVected by the Amazon/Orinoco Rivers, suggesting impediment to Accioly IV, Molina WF (2008) Cytogenetic studies in Brazilian subsequent dispersion and colonization by other Caribbean marine Sciaenidae and Sparidae Wshes (Perciformes). Genet Mol Res 7:358–370 species in Brazil. The second is cytogenetic, represented by AVonso PRAM, Galetti PM Jr (2005) Chromosomal diversiWcation of the conspicuous karyotypic diVerentiations currently Wxed reef Wshes from genus Centropyge (Perciformes, Pomacanthidae). in G. brasiliensis, which is much more likely to occur in the Genetica 123:227–233 123 Helgol Mar Res (2012) 66:363–370 369

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