Research Article |
Corresponding author: Maria Claudia Gross ( m.claudia.gross@gmail.com ) Academic editor: Irina Bakloushinskaya
© 2016 Renan Gabriel Gomes Junior, Carlos Henrique Schneider, Thatianna de Lira, Natália Dayane Moura Carvalho, Eliana Feldberg, Maria Nazareth Ferreira da Silva, Maria Claudia Gross.
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.
Citation:
Gomes Júnior RG, Schneider CH, Lira T, Carvalho NDM, Feldberg E, de Silva MNF, Gross MC (2016) Intense genomic reorganization in the genus Oecomys (Rodentia, Sigmodontinae): comparison between DNA barcoding and mapping of repetitive elements in three species of the Brazilian Amazon. Comparative Cytogenetics 10(3): 401-426. https://doi.org/10.3897/compcytogen.v10i3.8306
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Oecomys Thomas, 1906 is one of the most diverse and widely distributed genera within the tribe Oryzomyini. At least sixteen species in this genus have been described to date, but it is believed this genus contains undescribed species. Morphological, molecular and cytogenetic study has revealed an uncertain taxonomic status for several Oecomys species, suggesting the presence of a complex of species. The present work had the goal of contributing to the genetic characterization of the genus Oecomys in the Brazilian Amazon. Thirty specimens were collected from four locations in the Brazilian Amazon and three nominal species recognized: Oecomys auyantepui (Tate, 1939), O. bicolor (Tomes, 1860) and O. rutilus (Anthony, 1921). COI sequence analysis grouped O. auyantepui, O. bicolor and O. rutilus specimens into one, three and two clades, respectively, which is consistent with their geographic distribution. Cytogenetic data for O. auyantepui revealed the sympatric occurrence of two different diploid numbers, 2n=64/NFa=110 and 2n=66/NFa=114, suggesting polymorphism while O. bicolor exhibited 2n=80/NFa=142 and O. rutilus2n=54/NFa=90. The distribution of constitutive heterochromatin followed a species-specific pattern. Interspecific variation was evident in the chromosomal location and number of 18S rDNA loci. However, not all loci showed signs of activity. All three species displayed a similar pattern for 5S rDNA, with only one pair carrying this locus. Interstitial telomeric sites were found only in O. auyantepui. The data presented in this work reinforce intra- and interspecific variations observed in the diploid number of Oecomys species and indicate that chromosomal rearrangements have led to the appearance of different diploid numbers and karyotypic formulas.
Oryzomyini , FISH, telomere, rDNA, heterochromatin, COI
The order Rodentia is divided into nine taxonomic families in Brazil. The family Cricetidae contains the most members, among which the subfamily Sigmodontinae includes 86 genera and 395 species (sensu Reig 1980) according to
Currently, 16 species are recognized within this genus (
Karyotypes recorded for species of the genus Oecomys. Diploid Number (2n), fundamental number (FN) and location are listed.
Species | Location | 2n | FN | Reference |
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O. auyantepui | Jari river – PA | 72 | 80 |
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O. auyantepui | Jatapu river – AM | 64 66 | 110 114 | Present paper Present paper |
O. bahienses** | São Lourenço da Mata – PE | 60 | 62 |
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O. bicolor | Jari river – PA | 54 | 82 |
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O. bicolor | SUR | 80 | – |
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O. bicolor | RR Ipameri and Serra da mesa– GO | 80 | 124 |
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O. bicolor | Curanja river – PER | 80 | 134 |
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O. bicolor | Curanja river – PER | 80 | 136 |
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O. bicolor | Juruá river – AM | 80 | 140 |
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O. bicolor | Purus and Jatapu river – AM | 80 | 142 | Present paper |
O. bicolor | ? Hydropower plant UEH Samuel – GO | 82 | 110 |
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O. bicolor | Jari river – PA | 82 | 116 |
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O. bicolor | Jurua river – AM | 86 | 98 |
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O. catherinae | GO, São Lourenço da Mata – PE Ubatuba – SP, Cruz do Espírito Santo – PB, Igarassú, Jaqueira and Paudalho – PERJ | 60 | 62 |
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O. catherinae | Ubatuba – SPRJ | 60 | 64 | Pinheiro e Geise (2008) |
O. catherinae | RJ, SP | 86 | 98 |
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O. concolor | PAN | 58 | – |
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O. concolor | SUR | 60 | – |
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O. concolor | Villavicencio – COL | 60 | 62 |
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O. concolor | MEX | 60 | – | Andrade and Bonvicino (2003) |
O. concolor | MEX | 61 | – | Andrade and Bonvicino (2003) |
O. concolor | Curanja River – PER | 80 | 112 |
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O. concolor | DF, RJ, GO, SP, RO | 60 | 62 |
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O. paricola | Environment Park – PA | 68 | 72 |
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O. paricola | Marajó island – PA | 70 | 72 |
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O. paricola | Environment Park – PA | 70 | 76 |
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O. rex | Jari river – PA | 62 | 80 |
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O. roberti | AM | 80 | 114 |
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O. roberti | Juruá river – AM Jamari river – RO | 82 | 106 |
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O. rutilus | Negro river – AM | 54 | 90 | Present paper |
O. superans | PER Jurua river – AM | 80 | 108 |
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O. trinitatis | Jurua river – AM | 58 | 96 |
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Oecomys sp. | Cuieiras river – AM | 54 | 84 |
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Oecomys sp. | Jatapu – AM | 54 | 86 |
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Oecomys sp. | MS | 72 | 90 | Andrade and Bonvicino (2003) |
Hence, in the present study, we used classic and molecular cytogenetics approaches in order to enable the genetic characterization of three species of the genus Oecomys from the Brazilian Amazon. Further, we used DNA barcoding to evaluate the intra- and interspecific distances, and infer the utility in species identification by combining this dataset with sequences deposited in GenBank.
Thirty specimens were collected from five locations in the Brazilian Amazon (Fig.
Map of the Brazilian Amazon, indicating the collection sites. The left and right banks of the following Amazonas state rivers were sampled: 1 Jatapú (near the city of São Sebastião do Uatumã - 0°50’ to 1°55'S; 58°50’ to 60°10'W) 2 Negro (near the city of Santa Isabel do Rio Negro - 0°24.4'N; 65°1.017'W) 3 Purus (near the city of Tapauá - 05°42.183'S, 63°13.967'W) 4 Cuieiras (02°47'S, 60°27'W) 5 Tapajós (03°21.283'S, 55°11.733'W). BOL = Bolivia, PER = Peru, ECU = Ecuador, COL = Colombia, VEN = Venezuela, GUY = Guyana, SUR = Suriname, FRE = French Guyana, RR = Roraima, AP = Amapa, AM = Amazonas, PA = Para, RO = Rondonia, AC = Acre, MA = Maranhão, PI = Piaui, TO = Tocantins, BA = Bahia, MT = Mato Grosso, GO = Goias, MG = Minas Gerais.
Species of Oecomys collected in present work: The voucher, collection sites, sex, diploid number (2n), fundamental number (FN), karyotype formula, Nucleolus organizer region (NOR), rDNA 18S (18S), rDNA 5S (5s) are listed; M = male; F = female; m = metacentric; sm = submetacentric; st = subtelocentric; a = acrocentric; X = Sexual chromosome X; Y = Sexual chromosome Y. Bold voucher were karyotyped in the present work.
Species | Voucher | Sex | Collection sites | 2n | FN | Karyotype formula | NOR | 18S | 5S |
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O. auyantepui | INPA 6754 | M | Brazil, AM – Jatapú River | 64 | 110 | 12m+10sm+26st +16a+XY | 10p and 14p | 10p and 14p | 5p |
INPA 6751 | M | 66 | 112 | 16m+6sm+26st+ 14a+XY | |||||
INPA 6753 | M | – | – | – | – | – | – | ||
INPA 6747 | M | – | – | – | – | – | – | ||
O. bicolor | INPA 6772 | M | Brazil, AM – Purus River | 80 | 142 | 18m+10sm+36st+ 14a+XY | 15p, 18p, 21p, 22p and 26p | 2p, 3p, 13p, 15p, 16p, 18p, 19p, 21p, 22p, 25p 26p and 30p | 7p |
INPA 6749 | M | Brazil, AM – Jatapú River | |||||||
INPA 6756 | M | ||||||||
INPA 6758 | M | ||||||||
INPA 6757 | F | ||||||||
INPA 6752 | M | Brazil, AM – Jatapú River | – | – | – | – | – | – | |
INPA 6773 | F | Brazil, AM – Purus River | – | – | – | – | – | – | |
INPA 6770 | M | Brazil, AM – Negro River | – | – | – | – | – | – | |
INPA 6775 | M | Brazil, PA – Tapajós River | – | – | – | – | – | – | |
O. rutilus | INPA 6760 | F | Brazil, AM – Negro River | 54 | 90 | 24m+6sm+8st+ 14a+XX | 4p and 23p | 4p and 23p | 1p |
INPA 6761 | F | ||||||||
INPA 6762 | F | ||||||||
INPA 6768 | M | ||||||||
INPA 6767 | F | – | – | – | – | – | – | ||
INPA 6769 | M | – | – | – | – | – | – | ||
INPA 6766 | F | – | – | – | – | – | – | ||
INPA 6763 | F | – | – | – | – | – | – | ||
INPA 6764 | F | – | – | – | – | – | – | ||
INPA 6765 | F | – | – | – | – | – | – | ||
INPA 6774 | F | – | – | – | – | – | – | ||
INPA 6759 | F | – | – | – | – | – | – | ||
INPA 6745 | F | Brazil, AM – Cuieiras River | – | – | – | – | – | – | |
INPA 6746 | M | – | – | – | – | – | – | ||
INPA 6744 | F | – | – | – | – | – | – | ||
INPA 6750 | M | Brazil, AM – Jatapú River | – | – | – | – | – | – | |
INPA 6755 | M | – | – | – | – | – | – | ||
INPA 6748 | F | – | – | – | – | – | – |
Mitotic chromosomal preparations were obtained using the protocol described by Ford and Harmerton (1956), with some modifications. Nucleolus organizing regions (NORs), heterochromatin and G-banding were identified through silver nitrate staining (
Slides were screened for metaphases, at least 30 for each technique were analyzed and the best metaphases were photo-documented using an Olympus BX-51 epifluorescence microscope. Chromosomes were organized by decreasing size, and their morphology was determined based on the centromere position, being classified as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a) (
DNA was extracted according to the protocol described by
Sequences were manually aligned using BioEdit v7.2.2 software (
Two different diploid numbers were observed along the same bank of the Jatapú River: Karyomorph “a” exhibited 2n=64 chromosomes, a fundamental number = 110, and a karyotypic formula of 16m+6sm+26st+14a+XY (Fig.
Karyotypic characteristics of male Oecomys auyantepui, karyomorph “a” (INPA 6754) with 2n=64: a conventional Giemsa staining b heterochromatic regions highlighted by C-banding c G-banding d nucleolus organizing region-carrying pairs evidenced by silver nitrate staining e fluorescent in situ hybridization of 5S rDNA (green) and 18S rDNA (red) probes f karyotype indicating the presence of telomeric sites as well as interstitial telomeric sequence in the sex X chromosome. Bars: 10 µm.
Karyotypic characteristics of male Oecomys auyantepui karyomorph “b”, with 2n=66: a conventional Giemsa staining (INPA 6751) b heterochromatic regions highlighted by C-banding (INPA 6751) c G-banding (INPA 6751) d nucleolus organizing region-carrying pairs revealed by silver nitrate staining (INPA 6751) e fluorescent in situ hybridization of 5S rDNA (green) and 18S rDNA (red) probes (INPA 6751) f karyotype indicating the presence of telomeric sites as well as an interstitial telomeric sequence on the X sex chromosome (INPA 6754). Bars: 10 µm.
18S rDNA loci were visualized on chromosome pairs 10 and 14 of both karyomorphs, while the single 5S rDNA loci was located on pair 5 of karyomorph “a” and pair 7 of karyomorph “b” (Figs
Oecomys bicolor was found to exhibit a diploid number 2n=80 chromosomes, a fundamental number = 142, and a karyotypic formula of 18m+10sm+36st+14a+XX or XY, wherein pairs 12, 32, 33, 34, 35, 36, 37, 38 and 39 were metacentric; pairs 7, 20, 25, 26 and 27 were submetacentric; 1, 2, 5, 6, 10, 11, 13, 14, 15, 16, 17, 19, 21, 22, 23, 24, 29 and 30 were subtelocentric; and 3, 4, 8, 9, 18, 28 and 31 were acrocentric (Figs
Karyotypic characteristics of Oecomys bicolor: a conventional Giemsa staining of a male (INPA 6749) b highlighted sex chromosomes of a female (INPA 6749) c heterochromatic regions revealed by C-banding in a female (INPA 6772) d highlighted C-banding on a male’s sex chromosomes (INPA 6772) e G-banding of a female (INPA 6772) f nucleolus organizing region-carrying pairs revealed by silver nitrate staining (INPA 6749) g fluorescent in situ hybridization of 5S rDNA (green) and 18S rDNA (red) probes (INPA 6758) h karyotype indicating the presence of telomeric sites (INPA 6772). Bars: 10 µm.
Oecomys rutilus was characterized as showing a diploid number 2n=54 chromosomes and a fundamental number = 90, with a karyotypic formula 24m+6sm+8st+14a+XX or XY, in which pairs 3, 5, 11, 12, 13, 14, 15, 19, 21, 22, 25 and 26 were metacentric; 7, 8 and 20 were submetacentric; 1, 2, 4 and 6 were subtelocentric; and 9, 10, 16, 17, 18, 23 and 24 were acrocentric (Figs
Karyotypic characteristics of Oecomys bicolor: a conventional Giemsa staining of a male (INPA 6761) b highlighted sex chromosomes of a male (INPA 6768) c heterochromatic regions revealed by C-banding of a male individual (INPA 6754) d G-banding of a female (INPA 6761) e nucleolus organizing region-carrying pairs revealed by silver nitrate staining (INPA 6762) f fluorescent in situ hybridization of 5S rDNA (green) and 18S rDNA (red) probes (INPA 6761) g karyotype indicating the presence of telomeric sites (INPA 6761). Bars: 10 µm.
A total of 86 Oecomys mitochondrial COI gene sequences were compared: 33 originating from the present work and 53 deposited in GenBank (Appendix
The identification of Rodentia species is often difficult using morphological criteria alone (
The available chromosomal data for Oecomys species consist mostly of descriptions of diploid and fundamental numbers, which restricts comparisons with the data obtained in the present work (Table
Establishing the evolutionary direction of chromosomal rearrangements is not always possible because most of the available painting data for the Sigmodontinae group are incomplete, and it is not possible to draw definitive conclusions regarding the composition of a putative Sigmodontinae ancestral karyotype (
Both Oecomys auyantepui karyomorphs exhibit similar, predominantly centromeric, subtle heterochromatic blocks. Their NORs are also similar, with three different labeled sites being observed on the same chromosome pairs. The largest chromosomes of both karyomorphs are homologous - those carrying 5S rDNA loci in particular - sharing the same chromosomal region (subtelocentric chromosomes), position (long arm, proximal) and number of labeled sites, as inferred based on the increased resolution provided by G-banding. Thus, much like the NOR-carrying pairs, these chromosomes were not involved in chromosomal alteration processes leading to the occurrence of two different diploid numbers in O. auyantepui. Mitochondrial DNA analysis grouped all O. auyantepui specimens onto a single branch (Fig.
Current phylogenetic and karyotypic data suggest the existence of a complex of O. bicolor species (
The diploid number determined for O. rutilus (2n=54, first described in
Based on the amplitude of the genus distribution,
In the present work, NORs were found to be preferentially located in the terminal regions of chromosomes, and their number increased with the diploid number; this pattern is also present in other members of the family Cricetidae (
Oecomys species have undergone intense chromosomal alteration processes, as confirmed by the observed karyotypic patterns, indicating high local diversity and an ample distribution for the taxa under study. However, the limited taxonomic sample available, in terms of both Oecomys individuals and molecular data renders the determination of which evolutionary processes have led to the variability in karyotype morphology more difficult. Furthermore, the current data reinforce the necessity for integrative taxonomy, where genetic tools should be used in conjunction with morphological analysis to delimit Oecomys taxa.
The intra- and interspecific variations observed in the diploid number of Oecomys species indicate that chromosomal rearrangements such as fusions/fissions, translocations and duplications have led to the appearance of different diploid numbers and karyotypic formulas. However, telomere sequence hybridization was not found to be a good indicator of autosomal chromosome rearrangements in the Oecomys species under study, as no autosomal ITSs could be observed. O. bicolor, which is considered to be a derived taxon of the genus (
The authors thank the Chico Mendes Institute for Biodiversity Conservation [Instituto Chico Mendes de Conservação da Biodiversidade] for granting the license (10832-1 /35513-1), the Research Ethics Committee of the Federal University of Amazonas [Comitê de Ética em Pesquisa da Universidade Federal do Amazonas] for approving the field work (042/2013-CEEA/UFAM), and the SISBIOTA Amazônia team for performing the collections. Financial support was provided by the National Counsel for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq; 573976/2008-2, 558318/2009-6, 563348/2010-0), the Brazilian Federal Agency for the Support and Evaluation of Graduate Education [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES; Pro-Amazon Program: Biodiversity and Sustainability (Programa Pró-Amazônia: Biodiversidade E Sustentabilidade) Call no. 047 / 2012], and FAPEAM Edital P N. 020/2013. This study was supported by a graduate fellowship from CAPES to TLM and Research Productivity grants from CNPq to Eliana Feldberg.
COI sequences of Oecomys deposited in GenBank. The voucher, species and collection sites are listed.
Species | Genbank n° | Collection sites |
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O. auyantepui | JQ601277.1 | Suriname – Sipaliwini river |
JQ601224.1 | Suriname – Sipaliwini river | |
JQ601188.1 | Suriname – Kutari River | |
JQ601163.1 | Suriname – Kutari River | |
JQ601162.1 | Suriname – Kutari River | |
JQ601061.1 | Suriname: Brownsberg Nature Park | |
JQ601060.1 | Suriname: Brownsberg Nature Park | |
JQ601049.1 | Suriname: Brownsberg Nature Park | |
HQ545642.1 | Suriname: Sipaliwini River | |
HQ545608.1 | Suriname | |
HQ545608.1 | Suriname | |
JF491641.1 | Guiana: Upper Demerara-Berbice, West Pibiri, Mabura | |
JF491640.1 | Guiana: Upper Takutu-Upper Essequibo | |
JF491639.1 | Guiana: Upper Takutu-Upper Essequibo | |
JF491638.1 | Guiana: Potaro-Siparuni | |
JF491637.1 | Guiana: Potaro-Siparuni | |
JF491635.1 | Guiana: Cuyuni-Mazaruni | |
JF491634.1 | Guiana: Cuyuni-Mazaruni | |
O. bicolor | JQ601078.1 | Equador: Parque Nacional Yasuni |
JF491655.1 | Equador: Napo, Parque Nacional Yasuni | |
JF491652.1 | Equador: Orellana, Onkone Gare | |
JF491649.1 | Guiana: Demerara-Mahaic | |
JF491648.1 | Guiana: Barima-Waini | |
JF491647.1 | Guiana: Barima-Waini | |
JF491644.1 | Guiana: Upper Takutu-Upper Essequibo | |
JF491643.1 | Guiana: Potaro-Siparuni | |
JF491642.1 | Guiana: Potaro-Siparuni | |
JF444363.1 | Equador: Orellana | |
EU096822.1 | Suriname: Sipaliwini | |
EU096821.1 | Suriname: Sipaliwini | |
O. concolor | JF491662.1 | Equador: Napo, Parque Nacional Yasuni |
JF491661.1 | Equador: Orellana, Onkone Gare | |
JF444368.1 | Equador: Orellana | |
O. rex | JF491660.1 | Guiana: Potaro-Siparuni |
JF491659.1 | Guiana: Potaro-Siparuni | |
JQ601068.1 | Guiana: 40 Km NE of Surama | |
HQ919650.1 | Suriname | |
O. roberti | JF491663.1 | Guiana: Potaro-Siparuni |
O. rutilus | JQ601181.1 | Suriname: Kutari River Camp |
JF491682.1 | Guiana: Potaro-Siparuni | |
JF491681.1 | Guiana: Potaro-Siparuni | |
JF491673.1 | Guiana: Potaro-Siparuni Kabukalli Landing, Iwokrama Forest | |
JF491671.1 | Guiana: Siparuni river | |
JF491670.1 | Guiana: Barima-Waini, Baramita, Old World | |
JF491667.1 | Guiana: Barima-Waini, Baramita, Old World | |
JF491665.1 | Guiana: Upper Takutu-Upper Essequibo | |
EU095454.1 | Guiana: Upper Demerara-Berbice | |
EU095453.1 | Equador: Napo | |
Oecomys sp. | JF491619.1 | Equador: Napo, Parque Nacional Yasuni |