CompCytogen 12(3): 421-438 (2018) COMPARATIVE A reerrerewet open-access over doi: 10.3897/CompCytogen.v | 2i3.28736 Kan Cyto genetics http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics The karyotypes and evolution of ZZ/ZW sex chromosomes in the genus Characidium (Characiformes, Crenuchidae) Marcela Baer Pucci', Viviane Nogaroto’, Luiz Antonio Carlos Bertollo', Orlando Moreira-Filho', Marcelo Ricardo Vicari? | Departamento de Genética e Evolugéo, Universidade Federal de Sto Carlos, Rodovia Washington Luis, Km 235, 13565-905, Sto Carlos, Séo Paulo State, Brazil 2. Departamanento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Av. Carlos Cavalcanti, 4748, 84030-900, Ponta Grossa, Paranda State, Brazil Corresponding author: Marcelo Ricardo Vicari (vicarimr@uepg.br) Academic editor: /. Kuznetsova | Received 30 July 2018 | Accepted 7 September 2018 | Published 2 October 2018 http://zoobank.ore/D2BC52AE-7F91-49EE-970B-19B80B93A796 Citation: Pucci MB, Nogaroto V, Bertollo LAC, Moreira-Filho O, Vicari MR (2018) The karyotypes and evolution of ZZ/IZW sex chromosomes in the genus Characidium (Characiformes, Crenuchidae). Comparative Cytogenetics 12(3): 421-438. https://doi.org/10.3897/CompCytogen.v12i3.28736 Abstract Available data on cytotaxonomy of the genus Characidium Reinhardt, 1867, which contains the greatest number of species in the Characidiinae (Crenuchidae), with 64 species widely distributed throughout the Neotropical region, were summarized and reviewed. Most Characidium species have uniform diploid chromosome number (2n) = 50 and karyotype with 32 metacentric (m) and 18 submetacentric (sm) chromosomes. The maintenance of the 2n and karyotypic formula in Characidium implies that their genomes did not experience large chromosomal rearrangements during species diversification. In contrast, the internal chromosomal organization shows a dynamic differentiation among their genomes. Available data indicated the role of repeated DNA sequences in the chromosomal constitution of the Characidium species, particularly, in sex chromosome differentiation. Karyotypes of the most Characidium species ex- hibit a heteromorphic ZZ/ZW sex chromosome system. The W chromosome is characterized by high rates of repetitive DNA accumulation, including satellite, microsatellite, and transposable elements (TEs), with a varied degree of diversification among species. In the current review, the main Characidium cytoge- netic data are presented, highlighting the major features of its karyotype and sex chromosome evolution. Despite the conserved karyotypic macrostructure with prevalent 2n = 50 chromosomes in Characidium, herein we grouped the main cytogenetic information which led to chromosomal diversification in this Neotropical fish group. Copyright Marcela Baer Pucci et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 422 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) Keywords Chromosomal differentiation, Cryptic species, Repetitive DNA, Speciation genes Introduction Crenuchidae (Teleostei: Characiformes) include 18 genera and 95 species (Eschmeyer et al. 2018), grouped in Crenuchinae and Characidiinae (Buckup 1999). Characidium Reinhardt, 1867 is the most species-rich genus of Characidiinae, containing 64 valid species, which are morphologically very similar (Buckup 1993), and broadly distribut- ed across the Neotropical region (Eschmeyer et al. 2018). These fishes are small-sized, reaching 15 cm of length at adulthood (Buckup 1999), and some are commercially used in aquarium hobbies. They usually live in streams and can be found in both lentic and lotic habitats (Buckup 1999). Their elongated body shape and ventrally extended pectoral and pelvic fins enable them to attach tightly to the substrate, allowing them to resist to the water flow and capture food (Aranha et al. 2000). Characidium can be classified as autochthonous and insectivorous (Aranha et al. 2000, Bastos et al. 2013, Fernandes et al. 2017) and usually do not exhibit morphological sexual dimorphism (Buckup 1999). Characidium satoi Melo & Oyakawa, 2015 is an exception, where males develop a seasonal darker and uniform pigmentation of the body and head vs. the vertical bars exhibited in females (Melo and Oyakawa 2015). Phylogenetic analysis removed these fishes from the Characidae along with the Crenuchinae, and this group was organized in a new monophyletic family, the Cre- nuchidae (Buckup 1998). Phylogenetic relationships are available for most taxa in this family (Buckup 1993). According to available molecular and morphological data, Characidium is a monophyletic group, and its most recent common ancestor (Cre- nuchidae) likely originated during the Eocene, approximately 50.2 Mya. The geologi- cal events during this period boosted South American ichthyofauna diversity (Poveda- Martinez et al. 2016). Based on morphological data, Characidium zebra Eigenmann, 1909 is the most ancestral species of the genus as well as also of Characidiinae (Buckup 1993). An in- tegrative study using cytogenetic data combined to partial Cytochrome oxidase C subu- nit 1 (COD) and Cytochrome B sequences (Cyt B) for molecular phylogenetic analyses was applied in some Characidium species (Pansonato-Alves et al. 2014). This analysis proposed Characidium into two main groups of species: 7) those which do not exhibit sex chromosomes heteromorphism; and ii) those with a ZZ/ZW sex chromosome het- eromorphism with a partial or total heterochromatinization of the W chromosome (Pansonato-Alves et al. 2014). In addition, these data suggested: i) that the origin of sex chromosomes in analyzed Characidium species was unique and considered an apomorphic state and; ii) that B chromosomes present in some Characidium species presumably showed independent origins (Pansonato-Alves et al. 2014). Another common characteristic in cytogenetic data of Characidium is the occur- rence of cryptic species (Vicari et al. 2008, Machado et al. 2011, Pucci et al. 2014). This is suggested to be due to some populations of the same nominal taxa carrying the Chromosome evolution in Characidium 423 Z and W chromosomes at different stages of differentiation and apparent flow gene isolation (Vicari et al. 2008). Hence, new Characidium species are frequently described in the scientific literature (Melo and Oyakawa 2015, Zanata and Camelier 2015, Za- nata and Ohara 2015) and, the genus needs a critical revision. General chromosomal characteristics in Characidium Table 1 summarizes the recognized Characidium individuals/populations with cytogenet- ic data. ‘The first cytogenetic investigation of this genus was performed by Miyazawa and Galetti (1994), who analyzed four species and some populations of C. cf. zebra, Chara- cidium sp., Characidium cf. lagosantensis Travassos, 1947 and Characidium pterostictum Gomes, 1947, all of which had 2n = 50 chromosomes (Table 1). In fact, phylogenetically basal C. zebra, already possesses such chromosomal plesiomorphic features in the genus (2n = 50; 32m + 18sm), including the absence of heteromorphic sex chromosomes (Vi- cari et al. 2008, Machado et al. 2011, Pazian et al. 2013). This karyotype pattern occurs in most Characidium species (Table 1, Fig. 1), although rare spontaneous triploids have been detected among specimens of Characidium gomesi Travassos, 1956 (Centofante et al. 2001) and C. cf. zebra (Pansonato-Alves et al. 2011a). The evolutionary history of this genus revealed no large chromosomal rearrangements (Machado et al. 2011, Pucci et al. 2014, Scacchetti et al. 2015a, 2015b). However, occasional changes in the karyotypic formula can be found due to differences in the autosome morphology (Table 1). Interstitial telomeric sites (ITS), which are usually correlated with chromosomal fusions, were identified in the karyotypes of Characidium schubarti Travassos, 1955, Characidium lanei Travassos, 1967, Characidium lauroi Travassos, 1949, Characid- ium timbuiense Travassos, 1946, Characidium serrano Buckup & Reis, 1997, and two populations of C. pterostictum (Scacchetti et al. 2015c). The varied locations of ITS re- gions in the karyotypes were ascribed to their probable association with satellite DNA through transposition events and ectopic recombinations (Scacchetti et al. 2015c). Generally, the constitutive heterochromatin has a preferential distribution in the pericentromeric regions in the most Characidium chromosomes, but some large in- terstitial and terminal blocks were also observed. Chromosomal mapping of 18S and 5S rDNAs showed varied autosomal positions among Characidium genomes, ranging from single to multiple sites (Table 1). Nucleolar organizing regions (NORs) were probably related to the origin of the ZZ/ZW sex chromosome system that character- izes many Characidium species (Table 1), as commented below. Distribution of repetitive DNAs in the Characidium genome In fishes, tandem or dispersed repetitive DNA sequences are relevant markers for clari- fying karyotype evolution and sex chromosome differentiation (Schemberger et al. 2011, Barbosa et al. 2017, do Nascimento et al. 2018, Glugoski et al. 2018). Their 424 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) Table |. Review of Characidium cytogenetic studies until 2018. The variation in the diploid number (2n) is due to the presence of B chromosomes. “Unknown” signifies that the data was not available in the origi- nal study. NOR: Nucleolar Organizer Region; M: Metacentric; SM: Submetacentric; ST: Subtelocentric; A: Acrocentric. * The chromosome pairs are not indicated in the original publication. Sex chro- Species Localization 2n | mosome | Karyotype formula} rDNA18S rDNA5S References system Ribeirao Grande ; Centofante et al. C. alipioi Stream, SP. Brazil 50 | ZZ/ZW 30M+20SM Pair 16 (NOR) Unknown (2003) Travassos, 1955 | Ribeirao Grande . : Serrano et al. Sires 60! Biel 50-54} ZZ/ZW 32M+18SM Pair 18 Pair 20 (2017) C. fasciatum Bosse Reinhar dt, 1867 Francisco, MG, 50 | ZZ/ZW 32M+18SM Unknown Unknown Pazian et al. (2014) " Brazil Rio das Velhas C. cf. fasciatum | Stream, MG, 50 | ZZ/ZW Unknown Unknown Unknown Pazian et al. (2013) Brazil C. gomesi Paiol Grande 3 32 M+18 SM : Centofante et al. Travassos, 1956 | Stream, SP, Brazil Se ieee 2 31 M+19SM Pads eaiont (2001) C. gomesi (cited . ‘ like C cf Paranapanema, 50-54| ZZ/ZW 32M+18SM Three autossomic ake Maistro et al. ican SP, Brazil pairs* (1998) Pair 17 and : Pardo River, SP, an additional Maite ; 50-54} ZZ/ZW 32M+18SM Unknown (2004), Serrano ; Brazil chromosome * C. gomesi (NOR) et al. (2016) Machado River, 5 da Silva and MG, Brazil 50 Absent 32M+18SM Pair 17 (NOR) Unknown Maistro (2006) Quebra Perna 3 32 M+18 SM ae Stream, PR, 50 | ZZ/ZW pains Daranen Lg || Pr aL O08, Brazil 231M+18SM+1ST pair Pucci et al. (2014),. 32 M+18 SM Machado et al. Coe wétiesi Alambari Stream, . (2011) Pansonato- & SP. Brazil 50 | ZZ/ZW ZW Pairs 20 and 25 Alves et al. (2011b), 2 31 M+19SM Pazian et al. (2014) Novo River, SP, 3 32 M+18 SM ; ; Pansonato-Alves et Brazil 50-54} ZZ/ZW 231 Ma19SM Pair 18 Pair 25 al. (2011b, 2014) 3 32 M+18 SM Pairs 17, 22 and J in one of the C. gomesi a piven els 50 | ZZ/ZW homologous Unknown Mae ) fal Q31+18SM+1ST | of the pairs 1 and 20 Rio da Cachoeira ; StreameCO, so | zzizw | 32M+18SM Uhleiowh Ghanem en . oe , C. cf. gomesi Brazil Angra Brazil 50-52} ZZ/ZW 32M+18SM Unknown Unknown Pazian et al. (2014) Grande Rivee: Sl | 59° || 27/77.|| . 32M 185M Pair 17 Unknown | Machado eal Brazil (2011) Minhoca Stream, ; Machado et al. MG, Brazil 50 | ZZ/ZW 32M+18SM Pair 17 Unknown (2011) Tieté River, SP, Pansonato-Alves Brazil 50 | ZZ/ZW 32M+18SM ZW Unknown et al. (2014) C. gomesi Sao Domin: P Al g0s , ‘ ansonato-Alves River, MG, Brazil 50 ZZIZW 32M+18SM Pair 17 Unknown et al. (2014) Vermelho River, " Pansonato-Alves MT. Brazil 50 | ZZ/ZW 32M+18SM Pair 17 Unknown et al. (2014) 40 Jodo Ri 3 32 M+18 SM Sapijoao River, 50 | ZZ/ZW Pairs 10 and 17 Unknown Pucci et al. (2016) PR, Brazil 931M+18SM+1ST Chromosome evolution in Characidium 425 Sex chro- Species Localization 2n | mosome | Karyotype formula rDNA 18S rDNA 5S References system C. heirmostigmata da Graca & BarraGrande =| sy | zzizw | 32M+18SM Pair 4 Pair 19 Pucci et al. (2014) : River, PR, Brazil Pavanelli, 2008 C. lagosantens amendoiny ‘iiss *_ | Stream, MG, 50 | Absent Unknown Unknown Unknown __| Pazian et al. (2013) Travassos, 1947 f Brazil Cie. Infernao Lagoon, Miyazawa and Legussuiete SP. Brazil 50 | Unknown 32M+18SM Unknown Unknown Galetti (1994) Paros es 50 | ZZ/ZW | 32M+16SM+2A ZW Ode-autosomial” oleto et al.:(2009) PR, Brazil pair* C. lanei Pansonato-Alves Ti , 196 i ravassos, 1967 Cari Stream, PR, 50 | ZZ/Zw 32M+18SM ZW (NOR) One autospinal et al. (2010), Brazil pair Scacchetti et al. (2015b, c), S32 Me18 SM Centofante et al. i : . (2003) Pansonato- Cees Soran Puyen Sb 50 | ZZ/ZW ZW (NOR) Unknown | Alves et al. (2010), Travassos, 1949 | Brazil 231M+18SM+1ST Machado et al. (2011) C. oiticicai Pairaitinguinha Pansonato-Alves Travassos, 1967 | River, SP Brazil POD? AL SEM Teoh ZSTANORY Unknown et al. (2010, 2014) C. orientale .__ | Chasqueiro Pairs 1, 3, 5, 6, Scacchetti et al. seria? co Rey Stream, RS, Brazil DO eae oe EM 20 and W (2015a) Betari River, SP Pansonato-Alves Brazil 50-53) ZZ/ZW | 32M+16SM+2A ZW Unknown et al. (2010, 2014) Fati River, SP, Pansonato-Alves Brazil 50 | ZZ/ZW | 32M+16SM+2A ZW Unknown et al. (2014) Cari River, PR, Pansonato-Alves Brazil 50 | ZZ/ZW | 32M+16SM+2A ZW Unknown et al. (2014) Jacarei River, PR, Pansonato-Alves Brazil 50 | ZZ/ZW | 32M+16SM+2A ZW Unknown et al. (2014) ; Itapocu River, Pansonato-Alves C. pterostictum : 50 | ZZIZW | 32M+16SM+2A ZW Unknown SC, Brazil et al. (2014) Gomes, 1947 ae ee ooh airiquera-Acu airs 9, . River, SP. Brazil 50 | ZZ/ZW | 32M+16SM+2A ZW and 13 Pucci et al. (2014) Jacui River, RS, Three autosomal} Scacchetti et al. Brazil 50 | ZZ/ZW | 32M+16SM+2A ZW caine (2015b) Itapeva Lagoon, Scacchetti et al. RS, Brazil 50 ZZIZW | 32M+16SM+2A Unknown Unknown (20150) Carlos Botelho Mi d Ecological 50 | Unknown} 32M+16SM+2ST Unknown Unknown Bi tie a ; f Galetti (1994) Station, SP, Brazil C. rachovii Cabecas Stream, Pairs 1,3 ,5,17, | Scacchetti et al. Regan, 1913 RS, Brazil eee SZ eles gi 20 and W (2015a) Pansonato-Alves C. schubarti Cinco Réis River, et al. (2010), Travassos, 1955 | PR, Brazil PO ee eee poi elS) es Scacchetti et al. (2015c) C. serrano } : Biclap: 8 Ris. (oon ee so... ZZIZWE| SENEESNG DA Unknown Unknown Sees 1997 RJ, Brazil (2015c) C. stigmosum wey ‘ Mele Be Bideip, Sens BPE: | 50-1! athedne 32M+18SM Pair 23 pairs Le anda ee GO, Brazil (2015a) 2002 C. tenue (Cope, | Chui Stream, SC, : ; Scacchetti et al. 1894) Brazil 50 Absent 32M+18SM Pair 23 Pairs 1 and 7 (2015a) C. timbuiense _| Valsugana Velha Three autosomal) Scacchetti et al. Travassos, 1946 | Stream, ES, Brazil Bog) See) eh ue pairs* (2015b) 426 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) Sex chro- Species Localization 2n | mosome | Karyotype formula] rDNA18S rDNA5S References system C. vestigipinne ae : ‘ Buckup & aes Rives | so | zzizw | 32M+18SM ZW Seah Be ee y - Hahn, 2000 ana . ki C. vidali Bananeiras 50 | ZZ/ZW 32M+18SM One autosomal se oe eas Scacchetti et al. Travassos, 1967 | Stream, RJ, Brazil ij pair* thc e ‘. (2015b, c) autosomal pair sa Bananeiras ' Pairs 5, 12 Scacchetti et al. C. aff. vidali Sieaini, RJ; Bizzil 50-54| ZZ/ZW 32M+18SM Pair 21 nes (2015a) C. xavante da Graga, Pavanelli | Xingu River, MT, , . Scacchetti et al. & Buckup, Brazil 50 Absent 32M+18SM Pair 23 Pairs 1, 7 and 17 (2015a) 2008 C. zebra f : Pair 25 (NOR), r Eigenmann, cesar: 2 50 | Unknown 32M+18SM with 1 to 2 Unknown EERE 1909 i additional pairs : Miyazawa and Pasete hee Rive, Galetti (1994) Soe ate V5) 50 | Unknown 32M+18SM Pair 23 Pair 17 Machado et al. oh (2011), Pucci et al. (2014) Passa Cinco River, . 50-51 | Unknown Unknown Unknown Unknown Venere et al. (1999) SP, Brazil Piracicaba River, : Miyazawa and SP. Brazil 50 | Unknown 32M+18SM Pair 25 (NOR) Unknown Galetti (1994) Ribeirao Claro ; d 50 Absent Unknown Unknown Unknown Pazian et al. (2013) Stream, SP, Brazil Pansonato-Alves et Pairaitinga River, ; Pairs 1,6, and | al. (2010, 201 1a), SP, Brazil 50 Absent 32M +18SM Pair: 23 17 Scacchetti et al. (2015b, 2015c) C. cf. zebra Paiol Grand, Centofante et al. Segue E. 50 Absent 32M+18SM Pair 23 (NOR) Unknown (2001), Pucci et al. Stream, SP, Brazil (2016) Machado River, F da Silva and MG, Brazil 50 Absent 32M+18SM Pair 23 (NOR) Unknown Maistro (2006) Alambari River, ; 5 Pansonato-Alves SP. Brazil 50 Absent 32M+18SM Pair 23 Pair 17 et al. (201 1a) Novo River, SB é ' Pansonato-Alves Brazil 50 Absent 32M+18SM Pair 23 Pair 17 et al. (201 1a) Araqua River, SP, ; : Pansonato-Alves Brazil 50 Absent 32M+18SM Pair 23 Pair 17 et al. (201 1a) Duas Antas Scacchetti et al Stream, MT, 50 | Absent 32M+18SM Pair 23 Pairs land 17 | “@CCuM St ® : (2015a) Brazil Juba River, MT} ° . Pairs 1,6,9, 17 | Pansonato-Alves Brazil 50 Absent 32M+18SM Pair 23 Lace et al, (201 1a) Comedie. -9..leSUs4) SAbseht 32M+18SM | Pairs 4, 7 and 23 Pair 17 Pucci et al. (2014) Stream, SP, Brazil ida Sa GST Pairs 2, 4, 7, 20 rredeira airs 2, 4, 7, 20, ; . Stream, SP. Brazil 50 Absent 32M+18SM 23 and 17 Pair 17 Pucci et al. (2014) Preto River, SP Pansonato-Alves Brazil 50 | ZZ/ZW 32M+18SM ZW (NOR) Unknown et al. (2010) Characidium sp. | Lagoon of the Pairs 3, 7, 8, 23 Corredeira 50 | ZZ/ZW | 32M+16SM+2A ZW Mee Pucci et al. (2014) ‘ and 24 Stream, SP, Brazil Characidium Vermelho River, . ; Scacchetti et al. sp2 MT. Brazil 50 | ZZ/ZW 32M+18SM W and pair 7 Pair 17 (2015a) Hoenesa Ravens 1" Sou ZZIZMe | = SOME TESME Unlenown Wabnowa | Peaenee pls: » GO, Brazil 2014) Characidium sp. ane va rr nferno Lagoon, 7 iyazawa an SP. Brazil 50 | Unknown 32M+18SM Unknown Unknown Galetti (1994) Chromosome evolution in Characidium 427 Sex chro- Species Localization 2n | mosome | Karyotype formula] rDNA18S rDNA5S References system Characidium Russo River, MT; 50 | zzZ/zW 32M+18SM Pair 7 Pair 17 Scacchetti et al. sp.1 Brazil (2015a) Characidium Arinos River, MT, 50 | ZZ/ZW 32M+18SM Pair 1 Pair 1 Scacchetti et al. sp.3 Brazil (2015a) Characidium Nanay River, Peru} 50 ZZIZW 32M+18SM Pair 7 Pair 18 Scacchetti et al. sp.4 (2015a) Characidium Canoinha Stream, 4 ; Scacchetti et al. oS RS, Brazil 50 | ZZ/ZW 32M+18SM Pair 19 Pairs 1,5 and 6 (2015a) accumulation is a key factor for the morphogenesis and the differentiation process of sex chromosomes, and the induction of gene erosion (Matsunaga 2009, Schemberger et al. 2014, Ziemniczak et al. 2014). Despite the highly conserved karyotype structure, the genomes of Characidium species display a dynamic pattern of their internal chromosomal composition (Table 1, Fig. 2). Phylogenetics studies using mitochondrial DNA in Characidium were used to anchor a comparative cytogenetic analysis using telomeric DNA probe. This data indicated that the ITS signals found in genomes of some Characidium species (Fig. 2a) do not have relation with chromosome fusions but, on contrary, are associated with repetitive DNAs dispersion (Scacchetti et al. 2015c). Probably the ITS have origin in the evolutionary lineage of the genus in related hydrographic drainages (Scacchetti et al. 2015c), although some relationship species, such as C. zebra and C. gomesi, do not harbor such sequences. U2 small nuclear RNA (saRNA U2) had a highly conserved distribution in the first m pair in the most species (Fig. 2b), except for Characidium sp. aff. Characidium vidali Travassos, 1967, Characidium sp. 1 and Characidium alipioi Travassos, 1955, in which szRNA U2 site was located in the first submetacentric (sm) pair (Scacchetti et al. 2015b, Serrano et al. 2017). Distinct microsatellites also had a wide distribution in autosomal pairs (Fig. 2c), probably due to their association with TEs (Scacchetti et al. 2015b, Pucci et al. 2016), such as Tcl-Mariner (Fig. 2d). This pattern was also corroborated by Ser- rano et al. (2017), evidencing (CA),, and (GA), autosomal accumulation in the C. alipioi genome, as well as of several other microsatellites in C. zebra and C. gomesi. The molecular characterization and chromosome mapping of the histone genes H1, H3 and H4 were described for C. zebra and C. gomesi (Pucci et al. 2018). These three histone sequences appear to be associated with TEs and, iz situ localization, revealed that they are dispersed throughout the autosomes, but they are not in- volved in the differentiation of the specific region of the W sex chromosome in C. gomesi (Pucci et al. 2018). The available data point to the substantial role of repeated DNA sequences in the chromosomal constitution of Characidium species. However, due to the extension of the existing repetitive elements, additional investigations must address their signifi- cance in the evolutionary history of Characidium and, particularly, in sex chromo- some differentiation. 428 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) Characidium fasciatum (a) AMT tee an te axas A AR BA AX AY NK Ab Oe 14 HAuguvaenay ‘a (b) “fat ts W shy cf Pe Re ne 4 65 GF at 8 ‘¥ 1 ” Ay aL Wk OS Ue 9 @ <1 12 13 «48614 «615 16 EOD CMe ae oe an anne 17 18 19 20 21 22 23 24 25 Figure |. Representative karyotype of Characidium fasciatum with 2n = 50 chromosomes. Cytogenetic data revealed 32 m + 18 sm, without heteromorphic sex chromosomes: a conventionally Giemsa-stained b sequentially C-banded chromosomes. Scale bar: 5 um. Supernumerary and sex chromosomes in Characidium Several Neotropical fish species are carriers of supernumerary or B chromosomes (Car- valho et al. 2008). Additionally, due to the variety of simple or multiple sex chromo- some systems in these fishes, differentiated karyotypes exist between sexes (Moreira- Filho et al. 1993, Almeida-Toledo et al. 2001). B chromosomes, ranging from one to four chromosomes, were described in several Characidium species (Table 1). They are hypothesized to have different and independ- Chromosome evolution in Characidium 429 (a) C. lauroi - (TTAGGG)n (b) C. gomesi - U2 snRNA sm 17 18 19 20 (d) C. gomesi - Tc1/Mariner Figure 2. Fluorescence in situ hybridization using distinct classes of repeated DNA sequences as probes: In a karyotype of C. /auroi submitted to (TTAGGG) probing (red) b karyotype of C. gomesi evidencing U2 snRNA sites (red) € Karytype of C. heirmostigmata submitted to (GATA) | probing (red) and d karyo- type of C. gomesi evidencing Tcl/Mariner mapping (red). Scale bar: 10 pm. ent origins in evolutionary history of the species. To explain the origin, frequency and evolution of B chromosomes it was hypothesized that these elements are derivate from autosomes followed by gene silencing, heterochromatinization, and accumulation of repetitive DNA and transposons (Camacho et al. 2000, Vicari et al. 2011). In some species, B chromosomes are related to sex chromosomes due to share the same repeti- tive elements (Scacchetti et al. 2015a). In fact, genomes of C. gomesi, C. pterostictum and Characidium sp. aff. C. vidali displayed similar repetitive DNA sequences among B and sex chromosomes (Pansonato-Alves et al. 2014, Pazian et al. 2014, Scacchetti et al. 2015a, Serrano et al. 2016), while Characidium oiticicai Travassos, 1967 and C. alipioi did not show such shared sequences (Pansonato-Alves et al. 2014, Serrano et al. 2017, respectively). Despite their molecular homology, it was demonstrated that B and W chromosomes do not form multivalent pairings during meiosis in male and female C. gomesi individuals. Meiotic analyses revealed the bivalent pairing of the ZW chromosomes, as well as the bivalent plus one univalent formation in specimens carrying three B chromo- somes (Serrano et al. 2016). Chromosome pairing does not always indicate complete homology between chromosomes (Ramsey and Schemske 2002). In fact, the Z and W sex chromosomes in Characidium species possesses differences in 45S rDNA chromo- somal localization and in heterochromatin blocks extension (Fig. 3). Chromosomal localization differences of the repetitive sequences among Characidium species are also observed, such as in (T'TA),,, (GAG),,, (CG),, and (GATA)_, sequences (Scacchetti et al. 2015b, Pucci et al. 2016). In C. gomesi it was shown that the short arm of the W chromosome keeps homology with the terminal region of the Z chromosome in rela- tion to the (CG), ., (GATA) , and (TAA), sequences (Pucci et al. 2016). (GATA) and (TAA), homology is also present in the centromeric region of the C. gomesi (Pucci et al. 2016). These data help to explain ZW chromosome pairing and its bivalent forma- tion in Characidium species. 430 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) The occurrence of a ZZ/ZW sex chromosome system is another karyotypic char- acteristic of Characidium genomes. It was first described by Maistro et al. (1998) in Characidium cf. fasciatum Reinhardt, 1867 (Table 1), but it is also present in most Characidium species studied. The sex chromosomes in Characidium show a high degree of differentiation among species by chromosomal size, morphology, heterochroma- tin accumulation and presence or absence of rDNA sites (Maistro et al. 1998, 2004, Centofante et al. 2001, 2003, Vicari et al. 2008, Noleto et al. 2009, Pansonato-Alves et al. 2010, 2011b, 2014, Machado et al. 2011, Pazian et al. 2013, 2014, Pucci et al. 2014, 2016, Scacchetti et al. 2015a, 2015b, 2015c, Serrano et al. 2017), as exemplified in Fig. 3. Interestingly, the W chromosome can possess distinct cytotypes among C. gomesi populations, such as sm (Centofante et al. 2001, Pansonato-Alves et al. 201 1b) or subtelocentric (Vicari et al. 2008, Pucci et al. 2014, 2016). The majority of microsatellites sites were located in the terminal region of the Z chromosome and in the terminal/centromeric regions of W chromosome. The excep- tion is (I'TA),,, which was widely distributed throughout the whole W chromosome, and (GAG),,, which had a preferential accumulation in the W and B chromosomes of C. alipioi (Scacchetti et al. 2015b). (CG),, and (GATA) sequences were mainly found on the short arm of W chromosome in genomes of C. zebra and C. gomesi. It was suggested that these regions are enriched with sex-specific genes (Pucci et al. 2016), since the (GATA) sequences are known as a motif for sex- and tissue-specific GATA- binding proteins. However, this pattern was not found in Characidium heirmostigmata da Graca & Pavanelli, 2008 (Fig. 2). 18S rDNA sequences are also particular components of many Characidium sex chromosomes, occupying the short and the long arms of Z and W chromosomes, re- spectively, or the long arms of both sex chromosomes (Table 1, Fig. 3). These ribosomal sequences were likely associated with the origin of the protosex chromosome. It is likely that the NORs of the sm pair 23 (an ancestral pattern) were translocated to opposite arms of the second metacentric (m) pair (Machado et al. 2011, Pucci et al. 2014). Later differentiations in such protosex chromosomes were gradually acquired by isolated populations, leading to deletions and duplications in the rearranged regions due to meiotic pairing failures. Thus, recombination suppression mechanisms (re- arrangements, heterochromatinization, repeated DNA accumulation and gene ero- sion) were naturally selected, giving rise to distinct heteromorphic W chromosomes (Machado et al. 2011, Pucci et al. 2014). Such modifications also promoted the accu- mulation of the so-called “speciation genes”, particularly in linked Z chromosome loci (Pucci et al. 2014). These genes established meiotic barriers and post-zygotic isolation mechanisms, along with the morphological variations of W chromosome (Fig. 4). The current sympatric occurrence of some Characidium species does not display hybridization events among them. Sympatric and syntopic pairs of Characidium spe- cies, with the presence or absence of sex chromosomes, had already been described, namely C. alipioi and Characidium sp. cf. C. lauroi (Centofante et al. 2003), and C. cf. zebra and C. gomesi (da Silva and Maistro 2006). Thus, it is likely that NOR dis- placements throughout the genome was a key factor linked to W chromosome differ- Chromosome evolution in Characidium 431 A). pterostictum B) cgomesi ©) Cgomesi D) eal ZW Characidium sp. Characidium sp. Characidium spi Characidium sp3 Characidiumsp4 Characidium sp5 Characidium sp2 zeW zseW zseW z WwW z W Zz W Zz sW C. lanei C. lanei C.cf.gomesi C. gomesi C. alipioi C. gomesi z WwW z W zw z W z W zw C. lauroi C. lauroi C. orientale C. cf. fasciatum C. vidali z W z W z W z 6 z W C. oiticicai C. rachovii C. cf. gomesi C. cf. gomesi C. gomesi C. cf. gomesi C. gomesi | W 7 W PWS Fe Pe A Zw 42 C. pterostictum C. pterostictum C. pterostictum | C. timbuiense C. heirmostigamata z Ww z W z W zw z W C. schubarti C. vestigipinne C. oiticicai Zz sW zZeW zZ W Characidium sp. (9 Euchromatin aff. C. vidali @m Heterochromatin aa Telomeric region == Centromeric region GS W specific probe Gi 18S rDNA site “— 5S rDNA site z sW Figure 3. Idiograms showing main characteristics already identified for the ZZ/ZW sex chromosome system in Characidium species. It was highlighted the position of the centromere, distribution of euchro- matin and heterochromatin, W-specific probes, and rDNA sites. The a column detaches the species carry- ing 18S rDNA sites on the short and long arms of the Z and W chromosomes, respectively; the b column highlights the species bearing 18S rDNA sites on the long arms of both Z and W chromosomes; the c column shows the species that do not present 18S rDNA sequences on either Z or W chromosomes; the d column presents the species bearing Z and W chromosomes with unusual characteristics, including morphology, 18S and 5S rDNA sites, and W-specific probe distribution. 432 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) entiation in Crenuchidae. Usually, when the W chromosome is partially heterochro- matic, it is still a NOR bearing chromosome; but in totally heterochromatic chro- mosomes, NORs are found in different autosomes (Table 1, Fig. 3). Restriction-site associated DNA sequencing (RAD-seq) was applied to study the sex chromosomes of C. gomesi (Utsonomia et al. 2017). This application identifies 26 female-specific RAD loci, putatively located on the W chromosome, as well as 148 sex-associated SNPs showing significant differentiation. The use of W markers validated for in situ localization in other populations and species of the genus Characidium suggested a rapid turnover of W-specific repetitive elements (Utsonomia et al. 2017). This finding corroborates the inference that modifications on sex chromosomes also pro- mote the accumulation of the “speciation genes”, leading to chromosomal speciation mechanisms in Characidium. Perspectives on Characidium investigations Fish cytogenetic and molecular studies have improved over the last few years, especially with regard to better identification of the karyotypic evolution and sex chromosome dif- ferentiation among different groups of fish, as well as genes or specific regions related to sex determination. W-specific repetitive probes were already constructed for Characidium using microdissection from female metaphase chromosomes and degenerate oligonucleo- tide-primed PCR (DOP-PCR) or whole genome amplification (WGA) protocols. ‘These probes were later applied to chromosome painting in Characidium using a C. gomesi W- specific probe (Machado et al. 2011, Pazian et al. 2013, 2014, Pansonato-Alves et al. 2014, Pucci et al. 2014). This was followed by investigations of homologous regions between the sex pairs, B chromosomes and autosomes (Machado et al. 2011, Pazian et al. 2013, 2014, Pansonato-Alves et al. 2014, Pucci et al. 2014, 2016, Scacchetti et al. 2015a, 2015b, Serrano et al. 2016, 2017), and the cloning of a W-specific sequence that generated the CgW9 clone, which is similar to the zebrafish Helitron transposon (Pazian et al. 2014). The ZZ/ZW sex chromosome system is well-known and described. The repeated DNA classes related to gene erosion and differentiation of W chromosome, as well as regions or genes implicated in sex determination and gonadal differentiation, have not yet been properly investigated in most species. It has been demonstrated that the repeated DNA sequences are closely related to the regulatory genes network, particularly TEs, in a process called molecular co-option or exaptation (Feschotte 2008). In this sense, future studies concerning the dynamics of mobile elements and molecular co-option in the regu- latory system of Characidium will be relevant contributions to this research area. Sequenc- ing and comparisons between male and female genomes of different Characidium species will contribute to highlighting the genic and/or repetitive sequences that are sex-restricted. In other pathways, sequencing procedures of particular W fractions is needed for investigating specific genes related to sex determination and differentiation. Indeed, integrating cytogenetic, genomic, molecular, and bioinformatic tools will be essential for a better understanding of sex determination and differentiation processes in fishes, with applications in ecological and evolutionary studies. Chromosome evolution in Characidium 433 Protosex chromosome Allopatry Origin of the heteromorphic W sex chromosomes, Recombination with female sex-determining genes Supression mechanisms Rearrangements Repetitive DNA accumulation Heterochromatinization Gene erosion ; ZW Sucessive Vicariance events Independent evolution of ZZ/ZW sex chromosomes v Z chromosome accumulated the called speciation genes zw High differentiation degree Both Z and W sex chromosomes and morphologies of act as reproductive barrier W chromosomes in sympatric and syntopic pairs of species Figure 4. Schematic idiograms showing some steps proposed in the differentiation process of the ZZ/ ZW sex pair. The origin of the ZZ/ZW sex pair from the protosex chromosome of the Characidium spe- cies. Centromeric region (blue); 18S rDNA site (green); W specific probe region (red); probable Z specia- tion genes region (purple). Conclusion Chromosomal diversification in Characidium here revised show a diversified karyotype microstructure despite its conserved karyotypic macrostructure with prevalent 2n of 50 chromosomes arranged in 32 m + 18 sm. Differences in the number of rDNA sites, in heterochromatin blocks, in B chromosomes number and, in sex chromosomes sizes, as well as an interesting dynamic of repetitive DNAs on the chromosomes are observed among species, leading to chromosomal diversification and speciation. The data showed that different microsatellite expansions are involved in the sex chromo- some differentiation in Characidium. In addition, the microsatellite (T'TA),, play an important role in gene degeneration and erosion on the W chromosome in some Char- acidium species. These data are important for the molecular characterization of the W and B chromosomes, to karyotype structures determination and comprehension of cryptic species. Future studies integrating cytogenetic, genomic and molecular data open perspectives to understand the sex determination, B chromosome composition and, “speciation genes” in Characidium genomes. 434 Marcela Baer Pucci et al. / Comparative Cytogenetics 12(3): 421-438 (2018) Acknowledgements The authors are grateful to Instituto Chico Mendes de Conservagao da Biodiversidade (protocol number SISBIO 15117) for authorizing the capture of specimens. This study was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnolégico (CNPq), Coordenacéo de Aperfeigoamento de Pessoal de Nivel Superior (CAPES), Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Secretaria de Ci- éncia e Tecnologia do Estado do Parana (SETI), and Fundacao Araucaria de Apoio ao Desenvolvimento Cientifico e Tecnoldgico do Estado do Parana (Fundagao Araucaria). 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