Genetica (2011) 139:1499–1508 DOI 10.1007/s10709-012-9649-6 Differentiation of repetitive DNA sites and sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes) Michelle Orane Schemberger • Elisangela Bellafronte • Viviane Nogaroto • Mara Cristina Almeida • Guilherme Schnell Schühli • Roberto Ferreira Artoni Orlando Moreira-Filho • Marcelo Ricardo Vicari • Received: 11 September 2011 / Accepted: 9 April 2012 / Published online: 24 April 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Parodon and Apareiodon lack sufficiently consistent morphological traits to be considered a monophyletic group in Parodontidae. Species within this family are either sex-homomorphic or sex-heteromorphic (i.e., lacking a differentiated sex chromosome system, ZZ/ZW or ZZ/ ZW1W2). In this study, a DNA fragment from the heterochromatin segment of the W chromosome of Apareiodon ibitiensis (named WAp) was microdissected and used for in situ mapping of nine Parodontidae species. The species were also characterized using a satellite DNA probe (pPh2004). The species were phylogenetically clustered according to 17 characters, which were examined by both classical and molecular cytogenetic techniques. Given the present results, the single ZZ/ZW sex chromosome system seems to have been derived from a paracentric inversion of a terminal WAp site onto the proximal regions of the short arms of a Electronic supplementary material The online version of this article (doi:10.1007/s10709-012-9649-6) contains supplementary material, which is available to authorized users. M. O. Schemberger
V. Nogaroto
M. C. Almeida
R. F. Artoni
M. R. Vicari (&) Programa de Pós-Graduação em Biologia Evolutiva, Laboratório de Citogenética e Evolução, Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Av. Carlos Cavalcanti, 4748, Ponta Grossa, PR 84030-900, Brazil e-mail: vicarimr@pq.cnpq.br E. Bellafronte
O. Moreira-Filho Departamento de Genética e Evolução, Universidade Federal de São Carlos, Via Washington Luı́s-Km 235, São Carlos, SP 13565-905, Brazil G. S. Schühli Empresa Brasileira de Pesquisa Agropecuária-Embrapa Florestas, Estrada da Ribeira-Km 111, Caixa Postal 319, Colombo, PR 83411-000, Brazil metacentric chromosome pair, followed by WAp site amplification. We reason that these events restrained recombination and favored differentiation of the W chromosome in some species. Moreover, co-hybridization experiments targeting the WAp and pPh2004 repetitive DNA sites of A. affinis suggest that the ZZ/ZW1W2 sex chromosomes of this species may have arisen from a translocation between the proto-sex chromosome and an autosome. Our phylogenetic analysis corroborates the hypothesis of sex chromosome differentiation and establishes groups of closely related species. The phylogenetic reorganization in response to these new data supports the presence of internal monophyletic groups within Parodontidae. Keywords Chromosome microdissection
Cytosystematics
DOP-PCR
Dual color-FISH
Karyotype evolution Introduction Comparative cytogenetic studies have traditionally relied on the examination of chromosome banding patterns. However, when considering highly divergent species or species with highly rearranged genomes, cytogenetic comparisons of banding patterns are inadequate (Chowdhary and Raudsepp 2001). Hence, comparative chromosome painting localization associated with classical chromosomal markers has become the method of choice for performing cytogenetic genome comparisons. The advantage of such a method is that it allows for the identification of homologous genomic regions between species through cross-species in situ hybridization of whole chromosomes (Ráb et al. 2008; Vicari et al. 2010, 2011; Cabral-de-Mello et al. 2011a, b). Because these methods have been used to identify high levels of 123 1500 homology, some research groups now use chromosomal markers for phylogenetic population analyses (Freygang et al. 2004; Machado et al. 2011). Parodontidae is a small group of Neotropical fish with approximately 32 species divided into three genera: Parodon, Apareiodon and Saccodon. These genera have a wide geographical distribution throughout South America and part of Panama, except for some coastal basins and Patagonia (Pavanelli and Britski 2003). The taxonomy for Parodontidae is controversial because the family members lack diagnostic morphological traits reliable enough to support accurate phylogenetic analysis (Pavanelli and Britski 2003; Ingenito 2008). Ingenito (2008) argues that the existing morphological phylogenetic evidence for the genus Apareiodon is insufficient to support its continued maintenance; thus, Apareiodon could be regarded as a junior synonym of Parodon. The identification of new species in recent years suggests that Parodontidae has undergone population differentiation in the different South American hydrographic basins (Pavanelli and Britski 2003). These results were corroborated by cytogenetic analysis (Bellafronte et al. 2009, 2011). Cytogenetic studies have reported a conserved diploid number of 54 chromosomes in Parodontidae. However, Parodontidae species can be distinguished according to characteristics such as the presence of supernumerary chromosomes, distinctive karyotype formulae, numerical and position variation of pPh2004 satellite sequences, numbers of 18S and 5S rDNA sites, different heterochromatin patterns and the presence/absence of morphologically differentiated sex chromosomes (Bellafronte et al. 2011). Although numerous cytogenetic markers for Parodontidae exist, the spreading of the pPh2004 satellite DNA and sex chromosome differentiation are the major events known to lead to genome rearrangements (Vicente et al. 2003; Vicari et al. 2006; Bellafronte et al. 2009, 2011). Species of Parodon and Apareiodon containing or lacking the heteromorphic sex chromosome have both been observed. The cytogenetically analyzed species Apareiodon piracicabae, A. vittatus, Parodon pongoensis and P. nasus do not contain heteromorphic sex chromosome systems (Jesus and Moreira-Filho 2000a, b; Bellafronte et al. 2011). On the contrary, Parodontidae has been reported to have two sex chromosome systems: (1) a single ZZ/ZW system in P. hilarii, P. moreirai, A. vladii and A. ibitiensis and (2) a multiple ZZ/ZW1W2 sex chromosome system in A. affinis (Bellafronte et al. 2011). On the basis of these findings, Vicari et al. (2006) have proposed that the ZZ/ZW sex chromosomes of Parodon and Apareiodon share a common origin and that the separation of the genera Parodon and Apareiodon may in fact be artificial. However, little is known about the transformation process for the sex chromosomes of this family or their putative common origin. 123 Genetica (2011) 139:1499–1508 In the present study, to explore the sex chromosome differentiation model of Parodontidae, a DNA fragment from the heterochromatic segment of the W chromosome of A. ibitiensis (named WAp) was microdissected and subsequently amplified. The obtained W chromosomespecific probe was then used for in situ mapping on the chromosomes of nine Parodontidae species. Parodon hilarii satellite DNA (pPh2004, Vicente et al. 2003) was also used as a probe for the same species. The analyzed species differ in relation to the absence/presence of morphologically differentiated sex chromosomes and to the degree of sex chromosome differentiation. The obtained cytogenetic data were used to explore the karyotypic/ genomic divergence of Parodontidae, and the results indicated the existence of evolutionary chromosomal transformations of the heteromorphic sex chromosomes. Materials and methods Species analyzed and chromosome preparations Nine species from two genera of Parodontidae were selected for chromosome analysis: A. piracicabae, A. vittatus, A. ibitiensis, A. vladii, A. affinis, P. hilarii, P. moreirai, P. pongoensis and P. nasus (Table 1). Voucher specimens were deposited in the Zoology Museum of the Núcleo de Pesquisas em Limonologia, Ictiologia e Aquicultura of the Universidade Estadual de Maringá (Brazil). The procedures for sample collection were performed in compliance with the Ethics Committee on Animal Experimentation (process number: 04741/08) of the Universidade Estadual de Ponta Grossa (Brazil). Chromosome preparations were made from anterior kidney cells using an air drying method (Bertollo et al. 1978) and then processed for chromosome microdissection and fluorescence in situ hybridization (FISH). Repetitive DNA probes for FISH Two repetitive DNA probes were used for the chromosome mapping experiments: (1) a pPh2004 satellite DNA probe, isolated from the genomic DNA of P. hilarii (Vicente et al. 2003) and (2) a probe against the heterochromatic segment of the W chromosomes of A. ibitiensis, isolated in this work by microdissection of C-banded chromosomes (Vicari et al. 2010) and herein called WAp. Microdissection of the W chromosome of A. ibitiensis and DOP-PCR The W chromosomes of A. ibitiensis were microdissected from C-banded metaphase cells to ensure correct chromosome identification and the isolation of precisely the Genetica (2011) 139:1499–1508 1501 Table 1 Parodontidae species name, sampling locality, hydrographic basin name and number of specimens studied Species River (State) Hydrographic basin GPS localization A. piracicabae Rio Piumhi (MG) São Francisco -20°310 5500 and -46°020 4200 7 3 A. vittatus Rio Jordão (PR) Iguaçu -25°420 3100 and -51°530 5300 2 2 4 A. ibitiensis Rio Piumhi (MG) São Francisco -20°260 1600 and -45°550 3900 1 2 3 A. ibitiensis Rio Verde (PR) Alto Paraná -25°040 3500 and -50°040 0300 19 27 46 A. vladii Rio Piquiri (PR) Alto Paraná -25°010 4000 and -52°270 3200 13 19 32 A. affinis Rio Passa-Cinco (SP) Alto Paraná -22°250 2600 and -47°410 5600 7 5 12 P. hilarii Córrego do Porta (MG) São Francisco -17°210 1700 and -44°570 1500 11 14 25 P. moreirai Córrego Paiol Grande (SP) Alto Paraná -22°400 3400 and -45°410 0000 9 7 16 P. nasus Rio Paraguai (MT) Paraguai -15°340 4000 and -56°090 5800 6 4 10 4 5 9 P. pongoensis Rio Taquaralzinho (MT) Araguaia heterochromatin segment. The chromosome microdissections were performed using an inverted microscope (Olympus IX51) equipped with a mechanical micromanipulator (Narishige). The glass capillaries used for microdissection had a diameter of approximately 0.7 lm and were made using a micropipette puller (Narishige). Twelve C-banded W chromosomes were microdissected from A. ibitiensis. The heterochromatin segments from the W chromosomes were transferred to a microtube and used in the Degenerate Oligonucleotide Primed—Polymerase Chain Reaction (DOP-PCR). The W-probe was obtained by a DOP-PCR protocol (Telenius et al. 1992) adapted for the amplification of C-banded chromosomes (Vicari et al. 2010). The DOP-PCRs contained 19 ThermoSequenase reaction buffer, 40 lM dNTPs and 2 lM primer (50 ccg act cga gnn nnn nat gtg g 30 ). The reactions were heated at 95 °C for 10 min and then supplemented with 10 U of ThermoSequenase. The first amplification was performed by RAMP-PCR consisting of the following amplification parameters: 94 °C for 5 min; 12 cycles of low stringency annealing (94 °C for 1 min and 30 s, 32 °C for 2 min, a 0.2 °C/s temperature ramp up to 72 °C, followed by 72 °C for 2 min); and 35 cycles of high stringency annealing (94 °C for 1 min and 30 s, 52 °C for 1 min and 30 s and 72 °C for 1 min and 30 s). 0 00 No (#) 0 -15°53 28 and -52°14 56 00 No ($) Total 10 The WAp probe was digoxigenin-labeled by DOP-PCR. The WAp PCRs contained 100 ng of template DNA, 19 reaction buffer, 2 mM MgCl2, 40 lM dATP, dGTP and dCTP, 28 lM dTTP, 12 lM 11-dUTP-digoxigenin (Roche Applied Science), 2 lM DOP primer and 1 U Taq DNA polymerase (Invitrogen) and were subjected to the following amplification protocol: one cycle of 94 °C for 5 min; 35 cycles of 90 °C for 1 min and 30 s, 52 °C for 1 min and 30 s, and 72 °C for 1 min and 30 s; and one cycle of 72 °C for 5 min. FISH was performed under high stringency conditions (2.5 ng/lL probe, 50 % formamide, 29 SSC, 10 % dextran sulfate) according to the method of Pinkel et al. (1986). Signal detection was accomplished using an anti-streptavidin antibody conjugated to Alexa Fluor 488 (Invitrogen) and an anti-digoxigenin antibody conjugated to rhodamine (Roche Applied Science). The chromosomes were counterstained with DAPI (0.2 lg/mL) in Vectashield mounting medium (Vector) and analyzed using an Olympus BX41 epifluorescence microscope equipped with the DP71 digital image capture system (Olympus). The chromosomes were identified using the system proposed by Levan et al. (1964) and classified as either metacentric (m), submetacentric (sm) or subtelocentric (st). Phylogenetic analysis Fluorescence in situ hybridization FISH of the representative Parodontidae was performed using the pPh2004 and WAp satellite DNA probes. The cloned pPh2004 probe (Vicente et al. 2003) was labeled with biotin by PCR amplification using primers against the vector T7 promoter and M13 reverse sequences. The PCRs contained 20 ng of template DNA, 19 reaction buffer, 2 mM MgCl2, 40 lM dTTP, dGTP and dCTP, 20 lM dATP, 20 lM 14-dATP-biotin (Invitrogen), 0.3 lM of each primer and 1 U Taq DNA Polymerase (Invitrogen). The comparative analysis of discrete chromosome features (i.e., chromosome number and formulae, C-banding, and FISH results for the 18S and 5S rDNAs and the WAp and pPh2004 sites) reported in this study and from literature (references in Table 2) allowed for the construction of a data matrix of phylogenetic inferences for all of the Parodontidae species examined. The matrix contained mostly binary characters (15) and only two multistate characters (see Table 2). For each of the analyses, the Anostomidae, Leporinus friderici, was set as an outgroup to posterior ordination 123 1502 Genetica (2011) 139:1499–1508 Table 2 A summary of the seventeen characters (i.e., the presence/absence of the character, homologous chromosome and/or chromosome segments) used in the phylogenetics analysis Character State 0 State 1 State 2 State 3 References 1. 2n = 54 Absence Presence – – [1–16]; PS 2. Autosomal st chromosomes Absence Presence – – [1–16] 3. Pericentromeric heterochromatin on all chromosomes Absence Presence – – [1–16] 4. Terminal heterochromatin (number of chromosomes) Few: 0–17 Medium: 18–36. Many: [36 – [1–16] 5. Distal W euchromatin on the long arm Absence Presence – – [1–16] 6. Proximal W heterochromatin on the long arm Absence Presence – – [1–16] 7. pPh2004 sites on both terminal regions of one or more chromosome pairs Absence Presence – – [10, 11, 16]; PS 8. Sex chromosomes systems Absence Proto sex chromosome ZZ/ZW system ZZ/ZW1W2 system [1–16]; PS 9. Terminals WAp sites Absence Presence – – PS 10. Interstitial WAp sites on autosomes Absence Presence – – PS 11. Interstitial WAp sites on the sex chromosomes Absence Presence – – PS 12. Satellite DNA pPh2004 sites Absence Presence – – [10, 11, 16]; PS 13. Satellite DNA pPh2004 multiples sites Absence Presence – – [12, 13, 16]; PS 14. Satellite DNA pPh2004 sites on the sex chromosomes 15. 18S rDNA multiples sites Absence Absence Presence Presence – – – – [12, 13, 16]; PS [1–16] 16. 5S rDNA multiples sites Absence Presence – – [1–16] 17. Sinteny of the rDNAs Absence Presence – – [1–16] [1] Moreira-Filho et al. (1980); [2] Moreira-Filho et al. (1984); [3] Moreira-Filho et al. (1985); [4] Moreira-Filho et al. (1993); [5] Jesus et al. (1999); [6] Jorge and Moreira-Filho (2000); [7] Jesus and Moreira-Filho (2000a); [8] Jesus and Moreira-Filho (2000b); [9] Martins and Galetti (2001); [10] Centofante et al. (2002); [11] Vicente et al. (2003); [12] Bellafronte et al. (2005); [13] Vicari et al. (2006); [14] Rosa et al. (2006); [15] Bellafronte et al. (2009); [16] Bellafronte et al. (2011); PS present study of the character changes within Parodontidae. Unweighted parsimony (maximum parsimony, MP) analysis was conducted using PAUP*4.0b10 (Swofford 2002) and an exhaustive search that included 1,000 replicate random sequence addition searches and TBR branch swapping. The settings for this analysis were the following: unordered characters, steepest descent option not in effect, unlimited maximum tree number held in memory (auto-increased by 100), branches collapsed if maximum branch length is zero, and multiple trees option (MulTree) in effect. To explore the data further, the cladistic reliability of the characters (according to the level of homoplasy) was evaluated by successive approximation weighting (Farris 1969). The criterion for successive weighting was the default option in PAUP*4b10, the rescaled consistency index (Farris 1988). We estimated the branch support for the MP trees by nonparametric bootstrapping (Felsenstein 1985) using 1,000 replicate searches with simple addition sequences and TBR swapping. Results The cytogenetic features considered for the nine Parodontidae species are summarized in Table 2. DOP-PCR of 123 the C-banded W chromosomes of A. ibitiensis (Verde River, PR) yielded a smear of DNA species between 100 and 500 bp that was most intense approximately 250 bp. WAp probe efficiency to metaphase chromosomes from the Verde River A. ibitiensis species show complete labeling of the heterochromatin of the ZW chromosomes and additional sites at the terminal regions of some autosomes.1 These results confirm the extensive homology between the probe and the A. ibitiensis target. FISH with the WAp probe identified positive sites of homology at the terminal regions of some chromosomes in the karyotypes of all of the species tested (Figs. 1, 2). The A. piracicabae and A. vittatus karyotypes were characterized by 2n = 54 chromosomes (52 m/sm ? 2 st), and no sex chromosome heteromorphisms were verified by conventional staining or WAp probing (Fig. 1a, b, respectively). In A. piracicabae (Fig. 1a), the WAp probe sites were interspersed among nucleolar organizer regions (NORs—pair 27) in addition to several terminal sites on chromosomes. In contrast, A. vittatus (Fig. 1b) was found to have only terminal WAp sites. A. ibitiensis (Verde River) showed 2n = 54 chromosomes (48 m/sm ? 6 st to males and 47 m/sm ? 7 st to females) and a ZZ/ZW 1 Additional file 2: DOP-PCR product and WAp probe efficiency. Genetica (2011) 139:1499–1508 1503 Fig. 1 The karyotypes (as determined by WAp FISH) and respective collection sites of the Parodontidae species examined. The WAp signals are shown in red against the DAPI-counterstained chromosomes. In a A. piracicabae female (Piumhi River); b A. vittatus female (Iguaçu River); c A. ibitiensis male (Verde River); d A. ibitiensis female (Verde River); e A. ibitiensis female (Piumhi River); f A. vladii female (Piquiri River). The scale bar represents 5 lm heteromorphic sex chromosome system (Fig. 1c, d). The WAp probe detected the W chromosome heterochromatin heteromorphism, the proximal and terminal regions of the short arms of the Z chromosome and additional sites at the terminal regions of some autosomes (Fig. 1c, d). The karyotypes of A. ibitiensis (Piumhi River, Fig. 1e) and A. vladii (Fig. 1f) were organized in 2n = 54 chromosomes (50 m/sm ? 4 st) and a ZZ/ZW heteromorphic sex chromosome system. In these species/populations, the WAp probe detected homologous Z chromosome sites and minor WAp sequence content in W sm chromosomes compared to A. ibitiensis (Verde River). The males of A. ibitiensis (Fig. 1c), A. vladii, P. hilarii, P. moreirai, P. pongoensis and P. nasus (not shown) also demonstrated terminal and proximal WAp hybridization sites on the short arms of a metacentric chromosome pair corresponding to pair 10 in P. moreirai and pair 13 in the other species, designed proto-sex chromosome pairs of Parodontidae. The mapping of pPh2004 sites revealed that this satellite DNA sequence is only shared among P. pongoensis, P. nasus, P. moreirai, P. hilarii, and A. affinis (Fig. 2). Parodon pongoensis (Fig. 2a) shows 2n = 54 chromosomes (50 m/sm ? 4 st) and no heteromorphic sex chromosome system with conventional staining. However, FISH with the WAp probe demonstrated terminal and proximal WAp hybridization sites on the short arms of a metacentric chromosome pair corresponding to a proto-sex chromosome pair (Fig. 2a). Yet, P. pongoensis (Fig. 2e) was found to have pPh2004 marks on only one metacentric pair, at the terminal regions of the long arms of pair 13 (proto-sex chromosome). In P. nasus (Fig. 2b), the karyotype was organized by 2n = 54 chromosomes (48 m/sm ? 6 st), which is similar to an indiscriminate heteromorphic sex chromosome system in conventional staining and the presence of the proto-sex chromosome pair detected by WAp FISH. In this species, pPh2004 123 1504 Genetica (2011) 139:1499–1508 Fig. 2 The karyotypes of the Parodontidae species as determined by co-hybridization with the repetitive DNA probes WAp (red) and pPh2004 (green). In a P. pongoensis female (Taquaralzinho River); b P. nasus female (Paraguai River); c P. moreirai female (Paiol Grande River); d P. hilarii female (do Porta River); e A. affinis male and; f A. affinis female (Passa Cinco River). The scale bar represents 5 lm hybridization was observed at the terminal regions of the long arms of proto-sex chromosome plus three chromosomal pairs (Fig. 2b). Parodon moreirai (Fig. 2c) and P. hilarii (Fig. 2d) show 2n = 54 chromosomes (54 m/sm) and a differentiated ZZ/ZW sex chromosome. Parodon moreirai has a large m corresponding to a W chromosome, while in P. hilarii the W chromosome is a large st. In addition to detection of the Z chromosome, FISH with the WAp probe also revealed that the P. moreirai W chromosome (Fig. 2c) demonstrates a relative enrichment of the WAp target sequence, followed by a major WAp enrichment of the W chromosomes of P. hilarii (Fig. 2d). In P. moreirai (Fig. 2c), the pPh2004 sites were located at the terminal regions of the short arms of the W chromosome, the terminal region of the long arms of the Z chromosome, and occasionally at terminal positions on the long arms of an sm autosomal pair (pair 9). P. hilarii (Fig. 2d) displayed terminal signals on the short arms of the W chromosome, the long arms of the Z chromosome, and between 14 and 16 positions on autosomes. On the contrary, the A. affinis karyotypes were organized by 2n = 54 chromosomes (50 m/sm ? 4 st) in males (Fig. 2e), 2n = 55 chromosomes (49 m/sm ? 6 st) in females karyotypes (Fig. 2f) and a differentiated ZZ/ZW1W2 multiple sex chromosome system. The Z chromosome of A. affinis (Fig. 2e, f) displayed interstitial WAp sites on the long arms and terminal signals on both arms. WAp in this species also marked the W1 and W2 chromosomes and several terminal sites on autosomes. In this species (Fig. 2e, f), pPh2004 sites were mapped to terminal sites on both arms of metacentric chromosome pair 2 and to terminal sites on the long arms of metacentric pairs 5 and 23. Maximum parsimony analysis produced three minimum-length trees with 26-step length. Strict consensus for the obtained topologies resulted in a single tree with a consistency index (excluding uninformative characters) of 0.6800 and a retention index of 0.6923. The strict consensus was resolved, except for the tree polytomous nodes. 123 Genetica (2011) 139:1499–1508 1505 Fig. 3 Consensus tree generated by maximum parsimony with bootstrap values. The lines represent the characters related to the sex chromosomes responsible for the separation among groups. The letters represent the stage (branch) of karyotype differentiation. On the right side, idiogramatic representation of the sex chromosomes pair differentiation (red = WAp probe and green = pPh2004 probe) Computed bootstrap support values of greater than 50 % are presented in Fig. 3. Discussion Chromosomal marker analysis of Parodontidae has revealed remarkable interspecies diversity that has enabled researchers to make inferences about the process of genome diversification (Bellafronte et al. 2011). Several studies have reported the accumulation of repetitive DNA sequences during sex chromosome differentiation (Steinemann and Steinemann 2005; Martins 2007; Wang et al. 2009). While some species apparently lack differentiated sex chromosomes, our study shows that most Parodontidae species have either a proto-sex chromosome or a clear female heterogametic sex chromosome system. Most of these species appear to have undergone the same sex chromosome differentiation process, which involved an increase in the numbers of heterochromatic segments and the accumulation of repetitive DNA sequences on the W chromosome from an ancestral homomorphic pair. A similar situation is observed in the sister group Leporinus (Anostomidae), in which a ZZ/ZW sex chromosome system is shared by several species (Galetti et al. 1995; PariseMaltempi et al. 2007). The intragenomic movement of repetitive sequences might lead to faster chromosomal evolution (Wichman et al. 1991; Vicari et al. 2010). In Parodontidae, the distribution and expansion of pPh2004 satellite DNA seems to have a significant role in chromosomal diversification. Nonetheless, although this satellite DNA is present on sex chromosomes of some species, it seems to have no influence on rearrangements or differentiation of sex chromosomes in single sex systems. On the other hand, WAp repetitive DNA should be responsible for the differentiation of the ZZ/ZW sex chromosome system and, along with pPh2004 sequences, might be involved in the differentiation of the ZZ/ZW1W2 multiple sex system. Thus, the pathway of heteromorphic sex chromosome differentiation in Parodontidae is hypothesized. Evolution of sex chromosome systems in Parodontidae and phylogenetic consideration Evolutionary chromosome rearrangements can be considered ‘rare genomic changes’ (Rokas and Holland 2000) with very low levels of convergence. Hence, the observation of identifiable homologous chromosomes and chromosome segments represents a macroevent in an evolutionary lineage. Repetitive DNAs are often associated with genomic events involving duplication and spreading (Martins 2007), often precluding determinations of their evolutionary histories. The repetitive DNAs used for the inferences described here are from true homologous chromosomes, i.e., those identified on the unidirectional pathway of sex chromosome differentiation. The number of repetitive DNA sites is not included in our analysis because a fraction of these sites may be the result of intra-genome 123 1506 scattering events. The consensus tree presented in Fig. 3 summarizes the Parodontidae relationships. The result of this analysis shows that the hypothetical ancestor of Parodontidae is characterized by the following chromosomal features: 2n = 54 chromosomes m/sm, small amounts of proximal and terminal heterochromatin, terminal NOR in an m pair, the absence of pPh2004 satellite DNA and the absence of heteromorphic sex chromosomes. However, the features that are most important for understanding the chromosomal diversification of this group are the pPh2004 and WAp repetitive DNAs and the differentiation status of the heteromorphic sex chromosome systems. The WAp FISH experiments presented here have allowed us to determine that the A. piracicabae and A. vittatus species do not have heteromorphic sex. As for the differentiation of the sex chromosome pair, exclusively having terminal WAp sites seems to represent a simplesiomorphy in A. piracicabae and A. vittatus (Fig. 3, branch A). We find here that the metacentric chromosome pair corresponding to the modern Z chromosome, which we consider the proto-sex chromosome of Parodontidae, underwent an inversion event that rearranged the WAp sites to the proximal region of the short arms of these chromosomes (Fig. 3, branch B). Our data also indicate that this proto-sex chromosome underwent amplification of the WAp sites near the short arms, which led to chromosomal differentiation characterized by the accumulation of WAp sites on the long arms of the W chromosomes. Such a differentiation scheme was observed for A. ibitiensis and A. vladii (Fig. 3, branch C). The accumulation of WAp repetitive sequences corroborates the hypothesis regarding the derivation of the W chromosome in Parodontidae (Vicente et al. 2003). A similar phenomenon has been described for the amphibian Rana rugosa. Molecular analysis of the R. rugosa sex chromosome system was performed using the distribution pattern of the repetitive DNA sequence 31-REL and the structure of the ZW chromosome region containing this sequence. From these analyses, it was hypothesized that the divergence of the W chromosome was initiated by an inversion event that disrupted the repetitive DNA 31-REL sequence, leading to the subsequent accumulation of W-specific DNA on the proximal region (Suda et al. 2011). Ferreira and Martins (2008) also showed a predominant distribution of repetitive elements in the centromeric and telomeric regions and along the entire length of the largest chromosome pair (X and Y sex chromosomes) in the fish Oreochromis niloticus. Charlesworth et al. (2005) argue that data on young sex chromosomes are of great interest, particularly for addressing issues such as whether the sex-determining genes of an organism are initially on a single chromosome or whether the accumulation of repetitive sequences is the 123 Genetica (2011) 139:1499–1508 earliest event, before genes start degenerating. In some fish and plant species, the sex-determining genes have been genetically mapped to small regions of ordinary chromosomes, and some species show no sign of any extended sex chromosomal region. The current populations of A. ibitiensis and A. vladii have W chromosomes at different stages of differentiation (Rosa et al. 2006; Vicari et al. 2006; Bellafronte et al. 2009), which can be assessed according to the extent of accumulation of WAp sites on the W chromosomes and the number of rDNA sites (Fig. 3). In contrast, pPh2004 satellite sequences apparently exist on the proto-sex chromosomes of species from the genus Parodon. Therefore, once the species P. pongoensis, P. nasus, P. moreirai and P. hilarii bear pPh2004 satellite DNA at terminal sites on a metacentric pair comparable to the Z chromosomes in species with single sex chromosome systems, it is suggested that this undifferentiated chromosomal pair would be the origin center of such satellite DNA. In support of this interpretation, we note that the only species observed to have a pPh2004 satellite sequence on only the proto-sex chromosomes was P. pongoensis. Thus, these results indicate pPh2004 satellite DNAs originated in the terminal regions of the long arms of a pair of proto-sex chromosomes (Fig. 3, branch D), resulting in the grouping of species into a large clade composed of P. pongoensis, P. nasus, P. moreirai, P. hilarii and A. affinis (Fig. 3). We note, however, that the inclusion of A. affinis in this clade may be a misidentification. Parodon nasus and P. pongoensis satisfy the condition of having a pair of proto-sex chromosomes but present no differences in WAp sites on one homologue (W). These results support the argument that these species are homomorphic (Fig. 3, branch E). However, these species can be clearly distinguished by apomorphic synteny between the 18S and 5S rDNAs in P. nasus (Bellafronte et al. 2005). The amplification and accumulation of WAp sites proximal to the W chromosome appear to have been initiating events in the evolution of the sex chromosomes of P. moreirai and P. hilarii (Fig. 3, branch F). In addition to having sex chromosomes at different stages of WAp accumulation, P. moreirai and P. hilarii also have different numbers of pPh2004 satellite DNA sites (Fig. 2c, d, respectively). On the other hand, A. affinis shows a multiple sex chromosome system without evidence of W1 and W2 heterochromatin accumulation (Jesus et al. 1999). MoreiraFilho et al. (1980) have hypothesized that a centric fission of the first metacentric chromosome pair followed by pericentric inversion would explain the origin of the ZZ/ ZW1W2 sex system in A. affinis. Based on the identification of WAp sites on the Z, W1 and W2 chromosomes of A. affinis, it can be inferred that a translocation between autosomes and proto-sex chromosomes containing pPh2004 sites occurred during the evolution of multiple Genetica (2011) 139:1499–1508 sex chromosomes. In this model, the new combination of sex chromosomes would then undergo centric fission and further pericentric inversion to form the W1 and W2 chromosomes, as has been previously proposed by Moreira-Filho et al. (1980). However, species that have single sex chromosomes would retain the multiple sex chromosome system from an intermediate step in this process (White 1973). Multiple sex chromosome systems usually have small amounts of heterochromatin. Moreira-Filho et al. (1993) suggest that the amplification of heterochromatin from a single element (W) in highly differentiated single sex chromosome systems could prevent the formation of multiple sex chromosomes, as doing so would interfere with the balance of the meiotic trivalent. Alternatively, the presence of WAp repetitive DNA could facilitate the autosome proto-sex chromosome translocation as repetitive sequence may promote genome mobility (Wichman et al. 1991). In addition, the hypothesis of the Z, W1 and W2 sex chromosomes in A. affinis differentiating from an initial stage of single sex chromosomes in Parodontidae (i.e., proto-sex chromosomes or an initial W heteromorphism) is reinforced by the maximum parsimony branch (Fig. 3, branch G), which explains the apomorphic condition of A. affinis. Taxonomic implications The taxonomic validation of Parodon and Apareiodon within Parodontidae has been discussed during the last decade, although in the absence of substantial phylogenetic evidence to support their separation (Pavanelli and Britski 2003; Vicari et al. 2006; Ingenito 2008). The results of the chromosome-wide analysis described here suggest a possible pathway of chromosomal differentiation that challenges the current Parodontidae taxonomy. Consistent with the conclusions of Ingenito (2008), we find that the current delimitation of genera in this family may not reflect the natural groups. Nonetheless, our data do identify closely related species that can be organized into monophyletic clusters (Fig. 3). In conclusion, the results of this study suggest new hypotheses about the origin and diversification of chromosomal traits in the family Parodontidae. The mapping of repetitive WAp and pPh2004 sequences and the survey of conventional cytogenetic markers have allowed us to infer phylogenetic relationships within this family and trace the origin and differentiation of the sex chromosomes. This study illustrates that the WAp and pPh2004 repetitive DNAs are reliable evolutionary markers for Parodontidae and also key mediators of the remarkable karyotype diversification and speciation of this family. 1507 Acknowledgments The authors are grateful to Dr. Carla Simone Pavanelli for the identification of the specimens and to the Ministério do Meio Ambiente/Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (MMA/IBAMA—License number: 10538-1) for authorization to collect the biological samples. 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