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Neotropical Ichthyology

Print version ISSN 1679-6225On-line version ISSN 1982-0224

Neotrop. ichthyol. vol.16 no.1 Maringá  2018  Epub Mar 26, 2018

http://dx.doi.org/10.1590/1982-0224-20170135 

Original article

Development and characterization of fifteen polymorphic microsatellite loci in Bryconamericus aff. iheringii (Teleostei: Characidae) and cross-amplification in related Characidae species

Caroline Apolinário-Silva1 

Dhiego G. Ferreira2 

Analiza F. Cavenagh2 

Nícollas G. O. Aprígio2 

Bruno A. Galindo2 

Jens Carlsson3 

Silvia H. Sofia1 
http://orcid.org/0000-0002-3443-0696

1Laboratório de Genética e Ecologia Animal (LAGEA), Departamento de Biologia Geral, Universidade Estadual de Londrina, Rod. Celso Garcia Cid, km 380, 86051-970 Londrina, PR, Brazil. (CAS) carolapolinario07@gmail.com, (SHS) shsofiabelh@gmail.com, http://orcid.org/0000-0002-3443-0696 (corresponding author)

2Laboratório de Genética e Conservação (GECON)2, Universidade Estadual do Norte Paraná, Rua Portugal, 340 86300-000 Cornélio Procópio, PR, Brazil. (DGF) dhiegouenp@gmail.com, (AFC) chatcavenagh@hotmail.com, (NGOA) aprigio.bio@hotmail.com, (BAG) bruno@uenp.edu.br

3School of Biology and Environmental Science, UCD Science Education and Research Centre - West, University College Dublin Dublin 4, Ireland. jens.carlsson@ucd.ie


ABSTRACT

Data on 15 novel microsatellite loci from the Neotropical fish Bryconamericus aff. iheringii are presented here. Analyses of 32 individuals from four different streams revealed 192 different alleles, ranging from four to 32 alleles per locus (mean of 12.8 per locus). Observed and expected heterozygosities ranged from 0.094 to 0.813 and 0.205 to 0.952, respectively. These loci showed high polymorphic information content and will be a resource for genetic studies of B. aff. iheringii. Furthermore, several loci also amplified other small Neotropical Characidae (Piabarchus stramineus and Piabina argentea) and should be useful for these species.

Keywords: Genetic diversity; Lambarizinho; Molecular markers; Neotropical fish; SSR

RESUMO

Um total de quinze novos locos microssatélites é aqui apresentado para o pequeno peixe Neotropical Bryconamericus aff. iheringii. A análise de 32 indivíduos provenientes de quatro ribeirões diferentes revelou 192 alelos diferentes, variando de quatro a 32 alelos por loco (média de 12,8 por loco), e heterozigozidades observada e esperada variando de 0,094 a 0,813 e 0,205 a 0,952, respectivamente. O conjunto de locos obtido mostrou alto conteúdo de informação polimórfica e bom potencial para estudos genéticos de B. aff. iheringii, além disso vários locos amplificaram para outras espécies de pequenos Characidae neotropicais (Piabarchus stramineus and Piabina argentea).

Palavras-chave: Diversidade genética; Lambarizinho; Marcadores moleculares; Peixe Neotropical; SSR

Introduction

Bryconamericus Eigenmann, 1907 is one of the most diverse genera of the Characidae family, occurring in many river systems in of the Neotropical region, from Costa Rica to Argentina (Vari, Siebert, 1990; Lima et al., 2003). They are small omnivorous fish (not exceeding 10 cm in length) and inhabit a range of environments, including streams, rivers and lakes (Britski et al., 1988). Species of this genus are important food sources for piscivorous fish (Britski et al., 1988) throughout the range of environments, and in the Paraná River basin (one of the main drainage systems in South America) the genus is a prey item of several piscivorous fish (Hahn et al., 1997; Lowe-McConnell, 1999).

Bryconamericus aff. iheringii (Boulenger, 1887), regionally known as pequira or lambarizinho, is a member of this genus distributed in the upper reaches of the Paraná River basin (Graça, Pavanelli, 2007). Studies have suggested that B. aff. iheringii from the Paraná basin may constitute a distinct species from the B. iheringii found in the Laguna dos Patos system, Southern Brazil (Langeani et al., 2005, 2007). However, B. aff. iheringii populations are commonly found throughout the upper Paraná sub-basins, including rivers and small streams (Langeani et al., 2007; Costa et al., 2013; Cetra et al., 2016).

Fish less than 150 mm, such as B. aff. iheringii (maximum total length of 60 mm, Graça, Pavanelli, 2007), often dominate the ichthyofauna of Neotropical streams (Castro, 1999; Winemiller et al., 2008). The high prevalence of small fish dominating the ichthyofauna raises questions about the evolutionary and population aspects of these species. According to Castro (1999), the small size of these species usually contributes to a low movement capacity along the drainages. This reduced movement capacity could be one of the underlying explanations for the many endemic species and small-scale population structure found in Neotropical areas (Castro et al., 2003; Ferreira et al., 2016). However, genetic data for these species are largely lacking (Sofia et al., 2006, 2008; Ferreira et al., 2016). Biological data including genetic analyses of small fish, such as Bryconamericus spp., are essential for understanding biological and evolutionary properties, as well as for the management and conservation of the ichthyofauna of Neotropical streams (Agostinho et al., 2005; Dudgeon et al., 2006). Microsatellites are highly variable genetic markers suitable for cost-effective population genetic studies. However, particularly in the case of Neotropical fish species, the lack of suitable genetic markers has been one of the main obstacles to the development of more complete genetic studies involving this diverse group of fish. Thus, the present study aimed to identify and develop polymorphic microsatellite loci for Bryconamericus aff. iheringii and assess cross-species transferability to four other small Characidae from the Neotropical region.

Material and Methods

Microsatellite loci were obtained from an enriched genomic library using the methods described by Billotte et al. (1999) with some minor modifications (cf.Ferreira et al., 2013). The required genomic DNA was extracted from a sample of Bryconamericus aff. iheringii using the method of Almeida et al. (2001). Briefly, 5 μg was digested using 50 U of RsaI endonuclease and specific adapters RsaI-21 (5′CTCTTGCTTACGCGTGGACTA3′) and RsaI-25 (5′TAGTCCACGCGTAAGCAAGAGCACA3′) were linked to digested DNA using 5U of T4 DNA ligase (Invitrogen; www.invitrogen.com) in reaction buffer, including 10μM of each adapter in a final volume of 200μl and incubated at 20°C for 2 h. Fragments with putative microsatellite sequences were obtained using three probes bound to biotin: (AGA)5, (CT)8 and (GT)8. These fragments were amplified by Polymerase Chain Reaction and cloned into pGem-T Easy (Promega; www.promega.com) vectors using 5μl of amplification product, 50 ng of vector and 1 U of T4 DNA ligase in reaction buffer at 4°C (overnight). Cloning products were used to transform Escherichia coli (DH5- α lineage) cells.

A total of 96 positive clones were amplified using M13 primers (F and R) and subsequently sequenced using a Big Dye Terminator 3.1 sequencing kit (Applied Biosystems; www.appliedbiosystems.com) on an automated sequencer (ABI3500 xL, Applied Biosystems). BioEdit v.7.0 software (Hall, 1999) was used to visualize sequences and the Primer3 program (Rozen, Skaletsky, 2000) to generate microsatellite locus specific primers. Tests for potential hairpin structures and primer-dimers were conducted in AutoDimer software (Vallone, Butler, 2004).

Microsatellite validation was performed on a panel consisting of 32 individuals of B. aff. iheringii sampled from four streams (eight individuals per stream) in the Cinzas River basin (Grande - 23°16’00.70”S 50°27’58.06”W, Antas - 23°04’10.01”S 50°23’48.32”W, Barreiro- 23°38’44.97”S 50°23’32.32”W, and Bonito - 23°23’42.90”S 50°10’8.61”W streams), Southern Brazil. Cross-species amplification was trailed on extracted DNA from four other small Characidae species (five individuals per species), Piabarchus stramineus (Eigenmann, 1908), Piabina argentea Reinhardt, 1867, Serrapinnus notomelas (Eigenmann, 1915), and Hyphessobrycon eques (Steindachner, 1882), captured in the same drainages. Voucher specimens were deposited in the Museu de Zoologia da Universidade Estadual de Londrina (MZUEL) fish collection under catalog numbers, as follows: MZUEL 6457 (Bryconamericus aff. iheringii), MZUEL 6460 (Piabarchus stramineus), MZUEL 6466 (Piabina argentea), MZUEL 6467 (Serrapinnus notomelas) and MZUEL 6468 (Hyphessobrycon eques).

Microsatellite fragment length polymorphisms were analyzed on an automatic sequencer ABI PRISM 3500 xL, using GeneScan 600 Liz (Applied Biosystems) as standard molecular weight. Fluorescent labeling of fragments followed the method of Schuelke (2000), which includes an additional M13 sequence (5′- TGTAAAACGACGGCCAGT-3′) on the 5′ end of the forward primer.

PCR reactions were conducted in a total volume of 5μl, including 1 X GoTaq Master Mix (Promega), 10 ng of template DNA, 1% glycerol, 0.125 μM labeled M13 primer (either FAM, HEX or NED, Applied Biosystems, CA), 0.125 μM reverse primer, and 0.0125 μM forward primer. Amplifications were performed with an initial denaturation step at 94 °C for 4 min, followed by 35 cycles at 94°C for 40s, 48°C, 52°C, 56°C, or 60°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 30 min.

Genotyping was performed using the GeneMarkerv.1.85 program (SoftGenetics; www.softgenetics.com). Cervus v.3.0 (Marshall et al., 1998) was used to calculate the number of alleles (k), observed (H o ) and expected (H e ) heterozygosities, and polymorphic information content (PIC). The software package GENEPOP 4.0.10 (Raymond, Rousset, 1995; Rousset, 2008, using default settings) was used to test for conformance to the Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) and resulting probability values were corrected for multiple tests using the sequential Bonferroni correction (Rice, 1989). The genetic identity (I) and paternity exclusion (Q) probabilities (Paetkau et al., 1995; Weir, 1996) were estimated using Identity 1.0 (Wagner, Sefc, 1999, default settings). Endogamy coefficient (F IS ) was estimated using the program Arlequin 3.5 (Excoffier, Lischer, 2010). Tests (default settings) for the presence of null alleles and genotyping errors caused by large allele dropout and stutter were assessed using the programme MicroChecker v.2.2.3 (Van Oosterhout et al., 2004).

Results

Analyses of chromatograms for the 96 clones confirmed the presence of microsatellite repeats in a total of 46 sequenced clones. Based on sequence quality, microsatellite repeat structure, and primer suitability 15 loci (almost all dinucleotide repeats) were selected for further testing. These 15 loci all amplified successfully, showing polymorphisms that could be genotyped unambiguously. Information for these loci was submitted to the GenBank (accession numbers: MF693815-MF693829, Tab. 1).

A total of 192 alleles were detected across the 15 microsatellite loci (mean of 12.8 per locus), ranging from four (Bih26) to 32 (Bih16) alleles per locus (Tab. 1).

Tab. 1 Description of 15 polymorphic microsatellite loci isolated from Neotropical fish Bryconamericus aff. iheringii. Ta, optimal annealing temperatures; k, number of alleles; allele size range (bp); H o , observed heterozygosity; H e , expected heterozygosity estimated from 32 individuals; PIC, polymorphic information content; Q, paternity exclusion probability; I, probability of genetic identity; F IS , endogamy coefficient; GenBank accession numbers. *Significant value of endogamy coefficient (F IS ). 

Locus name Sequence repeat Primer sequences (5' - 3') Ta ( o C) k Allele size range (bp) H o H e PIC (Q) (I) F IS GenBank accession
Bih13 (CA)14 F: CGCTCATTCAAGGTGAGAGTC 56 22 184 - 258 0.719 0.933 0.929 0.864 0.285 0.244* MF693815
R: CTACGCCTTCCTACGCTGAG
Bih16 (GT)17 F: TAAGGAGAGCGTCAGAATCC 56 32 201 - 351 0.813 0.952 0.950 0.903 0.275 0.162* MF693816
R: CAGCAGCTCAGCTCTTCATC
Bih19 (AC)14 F: GGCATAAGGAATCCAGCATATC 56 24 169 - 243 0.781 0.930 0.926 0.858 0.287 0.175* MF693817
R: GTAACCGCTGATCACTAATAAGG
Bih22 (AG)13 GG (AG)3 F: GGTCCTGCACTCGCTGTTAG 52 19 211 - 281 0.375 0.853 0.840 0.852 0.336 0.570* MF693818
R: GCCAGCCAACGGTTAGAG
Bih26 (GT)4 A (AG)5 F: GAACTGCACGTTACATTCAGGA 52 4 175 - 197 0.250 0.514 0.465 0.285 0.658 0.524* MF693819
R: CTCAGCACTTCTGCCTGAC
Bih30 (TG)14 F: CTCCACGGCTGAAACAATG 48 7 159 - 187 0.414 0.601 0.551 0.440 0.493 0.327* MF693820
R: CCAGCGAATAATCAGTCTTCC
Bih31 (GT)14 F: GCATTGATATCCTGCCATCC 52 19 182 - 260 0.310 0.893 0.885 0.903 0.303 0.662* MF693821
R: TCTACCTGTCCATCGTGTCC
Bih40 (TG)2 C (TG)12 F: ACAGCGCATTCCTCCATTC 48 12 201 - 235 0.500 0.712 0.686 0.516 0.449 0.312* MF693822
R: AATCGACACGCTGCCAAG
Bih42 (AC)14 AA (AC)3 F: GAGCCGAACGCTGATGAC 52 7 108 - 174 0.094 0.205 0.201 0.113 1.121 0.552* MF693823
R: CCTCCAGACTTGACTACACCAC
Bih44 (GT)5 TAACACAG (GT)8 F: TCCTCTCATAGCGCTCTCTTC 48 6 138 - 166 0.500 0.730 0.693 0.510 0.429 0.329* MF693824
R: GCTTCACGTTACACCTGGAC
Bih45 (GA)6 (GT)14 F: CCGGATCAGCAGAACACAG 60 8 207 - 235 0.438 0.750 0.721 0.551 0.414 0.429* MF693825
R: CAGGTCAACTACAACGACACC
Bih47 (TTAT)5 (CT)9 F: CTCCTCCGATACCTGTGAACTC 52 7 126 -160 0.281 0.571 0.540 0.363 0.595 0.518* MF693826
R: CCAGCAGATACACAACACAGC
Bih73 (GT)17 F: CGCAAGCTTATGGTGAATCAG 48 9 220 - 268 0.188 0.427 0.416 0.267 0.779 0.571* MF693827
R: CAATGCACGTCATTCTGC
Bih76 (AC)19 F: CTGCATTACTCGGTAGGTTATATG 52 10 164 - 190 0.500 0.806 0.779 0.619 0.368 0.393* MF693828
R: CCTGTGCAATTAAGGAACTGTG
Bih77 (AC)8 (TG)2 TTA(AC)5 F: TGCCAACTTCATTAGAGCTTACAG 52 6 182 - 192 0.469 0.755 0.721 0.543 0.409 0.392* MF693829
R: CAGTTATATTCCTCACCTGATAGGAC
All loci 12.8 0.442 0.709 0.686 0.999 4.7x10-6 0.367*

The Bih42 locus showed the lowest observed (H o = 0.094) and expected (H e = 0.205) heterozygosities while the Bih16 showed the highest observed and expected heterozygosity (H o = 0.813 and H e = 0.952, respectively). The mean Polymorphic Information Content (PIC) for the 15 loci was 0.686. Following a scale proposed by Botstein et al. (1980), 12 loci (Bih13, Bih16, Bih19, Bih22, Bih30, Bih31, Bih40, Bih44, Bih45, Bih47, Bih76, and Bih77) were highly informative, with PIC values > 0.5, two loci (Bih26 and Bih73) were moderately informative (PIC > 0.25 and < 0.5), and only Bih42 showed low informative potential, PIC < 0.25 (Tab. 1). This set of loci demonstrated a very low value of genetic identity combined probability (4.7x10-6) and high combined probabilities of paternity exclusion (0.999), indicating high discriminatory power for population genetic studies.

Discussion

As expected, the mixture of individuals from different streams (possibly composing different populations) resulted in deviations from the Hardy-Weinberg equilibrium (HWE) and significant F IS values for all loci. This is likely an indication of a Wahlund effect caused by including elements of multiple genetic units in a single panel causing excess homozygosity and significant F IS estimates (Freeland, 2005). From the inflated homozygosity estimates, MicroChecker suggested null alleles were present at all loci. However, it is unlikely that the excess of homozygosity was caused by null alleles as it would require all analysed loci to be affected by null alleles. Instead we favour the hypothesis that the samples were derived from genetically distinct units and the pooling of these units caused a Wahlund effect. After correction for multiple tests (sequential Bonferroni correction, k= 105), thirteen loci combinations showed linkage disequilibrium, the locus Bih73 was linked to loci Bih30, Bih44, Bih45, Bih47and Bih77, the locus Bih45 linked to loci Bih31, Bih40 and Bih42, the locus Bih76 linked to loci Bih16 and Bih22, the locus Bih77 showed linkage with loci Bih30 and Bih44, and the locus Bih44 with Bih30.

The novel microsatellite loci developed in the present study constitute a highly variable marker set suitable for genetic studies of Bryconamericus aff. iheringii. Moreover, successful cross-species amplifications of some loci indicate that these microsatellites can be used for genetic studies of other small Characidae; Piabina argentea (12 polymorphic loci) and Piabarchus stramineus (nine polymorphic loci) (Tab. 2).

Tab. 2 Cross-amplification of the 15 polymorphic loci in four small Characidae species. N, sample size. The number in brackets represents the number of alleles. The superscript numbers are the number of individuals that successfully amplified per locus. - represents no amplification product. 

Primers Species N Microsatellite loci
Bih13 Bih16 Bih19 Bih22 Bih26 Bih30 Bih31 Bih40 Bih42 Bih44 Bih45 Bih47 Bih73 Bih76 Bih77
Allele size range (bp)
Piabarchus stramineus 5 162-172 202-216 156-168 208-220 - 179-187 - 210-238 - 163 225-229 132-164 219-225 - 190
(3)4 (5)5 (4)5 (3)4 - (2)5 - (4)5 - (1)5 (3)5 (6)5 (3)5 - (1)5
Piabina argentea 5 190-198 190-228 153-175 231-241 - 169-221 - 206-230 135-159 157-175 197-251 134-1508 212-244 172 185-199
(4)5 (5)5 (5)5 (3)5 - (6)5 - (5)5 (4)5 (7)5 (4)5 (4)5 (3)5 (1)5 (5)5
Serrapinnus notomelas 5 - 205-229 - 212 - 220-240 - 166-184 - - 195-223 - 262 - -
- (3)3 - (1)5 - (6)5 - (4)3 - - (2)5 - (1)5 - -
Hyphessobrycon eques 5 - - - - 203-237 - 220 131-133 - - - - - -
- - - - (8)5 - (1)5 (2)5 - - - - - -

Acknowledgments

We are grateful to the Universidade Estadual do Norte do Paraná (UENP) for financial and logistical support; Dr. Oscar Akio Shibatta (UEL) for his help in identifying the species studied and IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis), ICMBio-System (Instituto Chico Mendes-MMA) for granting permission to collect samples.

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Received: October 26, 2017; Accepted: February 12, 2018

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