, Volume 810, Issue 1, pp 227–237 | Cite as

Evolution in action: allopatry, variable diversity and a stepping-stone model of migration among populations of the freshwater bivalve Triplodon corrugatus from the north-eastern Amazon

  • Guilherme da Cruz Santos-Neto
  • Ismael Sander da Silva Nunes
  • Colin Robert Beasley
  • Adam Rick Bessa Silva
  • Cleidson Paiva Gomes
  • Claudia Helena TagliaroEmail author


Triplodon corrugatus is a freshwater bivalve (Hyriidae) endemic to the Amazon, Orinoco and Tocantins basins, and the Piriá river. Our understanding of hyriid diversity at, and below, the species level, remains poor. The genetic diversity of T. corrugatus from the Tapajós, Amazon, Tocantins, Irituia and Piriá rivers in the north-eastern Brazilian Amazon was investigated. Except for the Irituia, where a single COII–COI haplotype had been fixed, all the other populations had medium to high haplotype diversities, and all populations had low nucleotide diversities. Pairwise fixation indices indicated that all populations were structured, except for comparisons between the Tapajós and Amazon, and the Amazon and Tocantins rivers, which may be explained by a stepping-stone model of migration. AMOVA detected that 81.28% of the variation was among populations. However, STRUCTURE analyses corroborated only the Piriá river specimens as comprising a distinct population, which is being maintained by allopatry due to the current isolation between the Piriá, and the Amazon and Tocantins basins.


COII–COI Population genetics mtDNA Hyriidae Neotropical Conservation 


Triplodon corrugatus (Lamarck, 1819) is a South American freshwater bivalve, in the order Unionida, family Hyriidae Swainson, 1840, and tribe Hyriini (Graf & Cummings, 2007; Pereira et al., 2014), found in the Amazon–Orinoco basins (Pereira et al., 2014). Its shells and pearls have economic importance for the production of buttons, souvenirs and jewellery (Beasley, 2001; personal observation). Triplodon corrugatus, like other Hyriidae, has a larval stage, the glochidium, which is parasitic on freshwater fishes, resulting in the dispersal of juvenile mussels (Wächtler et al., 2001; Graf & Cummings, 2006; Pfeiffer & Graf, 2015).

In comparison to other regions of the world, including other parts of Brazil, there are relatively few studies of freshwater mussels in the Amazon (Pereira et al., 2014), and these usually deal with morphology and local occurrences (e.g. Bonetto, 1967; Haas, 1969; Mansur & Valer, 1992; Pimpão et al., 2012). Genetic studies, based on molecular data have been carried out on freshwater bivalves from Australia, North America, Europe and Africa, mainly to clarify systematic classifications (Graf et al., 2014, 2015; Santos-Neto et al., 2016; Pfeiffer & Graf, 2015; Lopes-Lima et al., 2017), but also to evaluate their population structure, variability and demography (Hughes et al., 2004; Mock et al., 2010; Jones et al., 2015; Froufe et al., 2014, 2016a, b; Inoue & Berg, 2017). Only a few genetic studies (Whelan et al., 2011; Graf et al., 2015; Pfeiffer & Graf, 2015; Santos-Neto et al., 2016; Combosch et al. 2017) have included Hyriidae from the Amazon region, all of which were used to establish phylogenetic relationships.

Whereas, molecular phylogenetics has dramatically improved our understanding of the higher level classification of the Hyriidae (Graf et al., 2015; Santos-Neto et al., 2016), our understanding of hyriid diversity at, and below, the species level, remains poor. Cytochrome c oxidase subunit I (COI) sequences have revealed interesting patterns of freshwater bivalve population structure, both within and among distinct basins, raising awareness of the conservation needs of specific populations (Hughes et al., 2004; Elderkin et al., 2007, 2008; Playford & Walker 2008; Mock et al., 2010; Froufe et al., 2014).

This is the first population genetics study of freshwater mussels from the Amazon region where we investigate if isolation by distance occurs among populations of T. corrugatus from the Amazon and Tocantins basins and whether a geographically isolated population in the Piriá river is distinct from the rest.

Materials and methods


A total of 158 specimens of Triplodon corrugatus were manually collected at depths of up to 3 metres from five rivers (Table 1; Fig. 1): Tapajós and Amazon, both in the eastern part of the central Amazon basin; Tocantins, further east and connected to the Amazon river via the Pará river; Irituia, which flows into the Guamá river, and which then enters the Tocantins river; and the Piriá river, which is furthest east and is not connected with any of the others, flowing directly into the Atlantic Ocean, approximately 500 km east of the mouth of the Amazon.
Table 1

Summary of information on haplotypes found in samples of Triplodon corrugatus from five rivers from the north-eastern part of the Amazon region of Brazil, including sample size (N), geographic coordinates (decimal degrees) of the sampling sites, total number of haplotypes (H), and haplotype (h) and nucleotide (π) diversities for each river



Sampling site coordinates (Longitude, Latitude)






−54.916000, −2.478070






−54.010506, −1.981249






−49.413628, −2.455167






−47.433396, −1.774542






−46.469513, −1.426549




Fig. 1

Study area in South America (rectangle, inset) showing the north-eastern part of the Amazon region of Brazil with locations on the Tapajós, Amazon, Tocantins, Irituia and Piriá rivers where populations of Triplodon corrugatus were sampled

Many bivalves have doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA), involving distinct male (mtDNA M) and female (mtDNA F) lineages (for details, see Zouros, 2013). Curole & Kocher (2002) detected a 200-codon extension of the COII gene in the mtDNA M when compared with the mtDNA F in Unionidae. In order to check for an extension of the COII gene in mtDNA M of T. corrugatus, sex was determined in all specimens collected in the Piriá, Irituia and Tocantins rivers by examining fresh gonad smears under the light microscope (400×).

Laboratory methods

The bivalves were stored in 92% ethanol at −20°C and total genomic DNA was extracted from the adductor muscle. Samples were kept in proteinase K at 37°C overnight and the extraction procedure followed the phenol–chloroform protocol of Sambrook et al. (1989).

DNA amplification of the region, including part of the cytochrome c oxidase subunit II gene, a spacer region, and part of the cytochrome c oxidase subunit I (COII–COI), was carried out using the following pair of primers: UNIOCOII_2 (Curole & Kocher, 2002) and HCOC2198 (Folmer et al., 1994). The polymerase chain reaction (PCR) consisted of 4 μl of dNTP (1.25 mM), 2.5 μl of buffer (10X concentrated), 0.75 μl of MgCl2 (25 mM), 0.5 μl of each primer (200 ng/ml), 1–1.5 μl of total DNA (200 ng), 0.5 μl of Taq DNA polymerase (5 U/μl) and distilled water to complete the final reaction volume of 25 μl. The amplification protocol consisted of an initial denaturing at 94°C for 3 min, followed by 40 cycles at 94°C for 1 min (denaturing), 50°C for 1 min (annealing), 72°C for 2 min (extension), and a final extension period of 7 min at 72°C. Sequences were obtained using automatic sequencers (MegaBace 750; Applied Biosystems 3500 XL), following the manufacturer’s protocols.

Genetic analyses

COII–COI sequences (760 sites) from 158 specimens of Triplodon corrugatus were aligned using ClustalW (Thompson et al., 1994) implemented in BioEdit 7.2.5 (Hall, 1999), after which, minor corrections in the alignments were carried out. All sequences were analysed in the forward direction and were used in the following analyses. Mega 6.06 (Tamura et al., 2013) was used to verify variable nucleotide and amino acids sites, as well as termination codons. Haplotype (h) and nucleotide (π) diversities (Nei, 1987) of each population were estimated in Arlequin (Excoffier & Lischer, 2010). The Arlequin project file was generated using DNASP 5.1 (Librado & Rozas, 2009). The haplotype networks were designed with Haploviewer (Salzburger et al., 2011), in order to establish the relationship between different haplotypes, considering only variable sites using parsimony algorithms. An extra network based on COI (494 sites) was designed in order to compare the north-eastern Amazon haplotypes with two sequences of T. corrugatus from Peru (GenBank: JN243890; KP184900).

Tajima’s (D; Tajima, 1989) and Fu’s (Fs; Fu, 1997) tests were used to verify if the populations were in a state of selective neutrality, and to infer the demographic history of the populations. Both Tajima’s and Fu’s tests were run in Arlequin (Excoffier & Lischer 2010) and the interpretations were based on Tajima (1989, 1993) and Fu (1997). The mismatch distribution (number of observed differences between haplotypes of pairs of samples) was used to evaluate the demographic history of the T. corrugatus populations. This analysis allows identification of populations that were stable, or have passed through bottlenecks in the past, or are in expansion (Frankham et al., 2010). The sum of the squared deviations (SSD) between observed and expected mismatch distributions were calculated in Arlequin (Excoffier & Lischer, 2010).

A Bayesian cluster method performed in STRUCTURE 2.2.3 (Pritchard et al., 2000) was used to measure the possibility of genetic clustering among all the samples (N = 158) collected at the five sites. The range of possible clusters (K) tested was set from 2 to 8, and 10 independent runs were carried out for each, using no prior information, and assuming admixture and correlated allele frequencies. Lengths of Monte Carlo Markov Chain (MCMC) interaction and burn in were set at 106 and 104, respectively. The correct K number of clusters was defined using the highest value of the log likelihood [Ln Pr(X/K)] of the data posterior probability for a given K (Pritchard et al., 2000). The Δ(K) statistic, based on the second-order rate of log probability of the data (Evanno et al., 2005), was analysed using the on-line program STRUCTURE HARVESTER 0.0.91 (Earl & vonHoldt, 2012) in order to estimate the appropriate K value. Genetic structure among populations was also tested by pairwise Fst (Wright, 1965). The distribution of genetic variability within and between populations was inferred from an Analysis of Molecular Variance (AMOVA, Excoffier & Lischer, 2010) for all five rivers, performed with 10,000 permutations in Arlequin (Excoffier & Lischer, 2010).

The pairwise correlation among Fst and geographical distances in km (Table 2), using the shortest distance along the river channels among the five Amazonian populations (Tapajós, Amazon, Tocantins, Irituia and Piriá) was calculated, using 10,000 permutations, with IBDWS 3.23 (Jensen et al., 2005).
Table 2

Pairwise comparison of the fixation index (Fst) with corresponding probabilities (P; lower diagonal) among samples of Triplodon corrugatus from five rivers in the north-eastern part of the Amazon region of Brazil, based on COII–COI sequences and estimates of their geographic distances along river channels (km; upper diagonal)














P = 0.661






P = 0.025*


P = 0.052





P < 0.001*


P < 0.001*


P < 0.001*




P < 0.001*


P < 0.001*


P < 0.001*


P < 0.001*

* P < 0.05 is significant

a Geographic distance assuming an ancient connection via the Guamá River

Phylogenetic analyses were performed with PhyML 3.1 (Guindon & Gascuel, 2003) for the maximum likelihood (ML) method and MrBayes 3.2 (Ronquist et al., 2012) for a Bayesian analysis (BA). T. corrugatus from Peru (JN243890; KP184900) and Paxyodon syrmatophorus (KU888254, KU888255) were included in the database. Castalia ambigua (KU888242), Castalia stevensi (AF231736) and Callonaia duprei (KU888234) were used as an outgroup. A saturation test was performed using DAMBE 5 (Xia, 2013). jModelTest 2.1.7 (Darriba et al., 2012) was used to choose the best model for use in the ML and BA analyses, computing the likelihood scores and using the Akaike Information Criterion correction (AICc) and Bayesian Information Criterion (BIC), respectively, (Darriba et al., 2012). The best choice of model was the Hasegawa–Kishino–Yano, HKY (Hasegawa et al., 1985) for ML and for BA. The jModelTest commands used were: AICc and BIC: “Lset base = (0.1601 0.0986 0.3129) nst = 2 tratio = 4.6253 rates = gamma shape = 0.1570 ncat = 4 pinvar = 0”.


Reliable alignments from COII to COI yielded a total of 760 sites. All male and female T. corrugatus were found to share the same haplotypes in their matrilinear COII–COI sequences amplified from adductor muscle, thus eliminating the possibility of having amplified the patrilinear sequences due to doubly uniparental inheritance. Considering all five populations, a total of 37 haplotypes were identified (Fig. 2) and the sequences were deposited in GenBank (MF441556 to MF441592). The most frequent being: H1 in the Irituia river; H2 in the Tocantins, Tapajós and Amazon rivers; and H17 in the Piriá river. The H1 haplotype was the only one identified from the Irituia river population, although it was also found at a low frequency (0.06) in the Tocantins river. The Tapajós, Amazon and Tocantins populations shared two haplotypes (H2, H7), and the Tapajós and Amazon also shared H27. All haplotypes from the Piriá river were private. The network (Fig. 2) divided the COII–COI haplotypes into three haplogroups: H17 to H22 (Piriá river), H4 and H6 (Tocantins river) and all the other haplotypes (Tapajós-Amazon-Tocantins-Irituia rivers), with at least seven mutations separating the Piriá samples from the others. Except for the Irituia River, all the other populations had medium to high haplotype diversity, whereas all of them displayed low nucleotide diversity (Table 1). Translation of the COI nucleotide sequences showed that all Piriá haplotypes had a private amino acid substitution, and a second one shared only with H4 and H6 from the Tocantins. A termination codon was observed at the end of each of the COII gene sequences.
Fig. 2

Haplotype networks estimated from Maximum Parsimony analysis of samples of Triplodon corrugatus from the Tapajós, Amazon, Tocantins, Irituia, and Piriá rivers in the north-eastern Brazilian Amazon and two samples from Peru (inset)

Tajima’s (D) and Fu’s (Fs) neutrality tests (Table 3) showed negative and significant values for both tests in the Tapajós, Amazon and Tocantins populations. The comparisons between the observed and the expected mismatch for each of these three populations were not rejected by the sum of squared deviations (SSD; Table 3). The haplotype H1 was fixed in the Irituia population, probably due to a recent bottleneck. The Piriá population neutrality test results were consistent with neutrality. The pairwise fixation indices (Fst) and their probabilities (P), based on COII–COI sequences, indicated that all populations were structured, except for the comparisons between the Tapajós and Amazon rivers (Fst = −0.00619; P = 0.661), and between the Amazon and Tocantins (Fst = 0.01838; P = 0.052). AMOVA detected that 81.28% of the variation was among the heterogeneous populations (P < 0.001). The correlation between genetic (Fst) and geographic distances (km) among the Tapajós, Amazon, Tocantins, Irituia and Piriá sites was not significant (Z = 3972.9, r = 0.21, P = 0.1182, see Fig. 3).
Table 3

Summary of the neutrality tests of Tajima (D) and Fu (Fs), sum of the squared deviations (SSD) and their corresponding probabilities (P), based on sequence data of populations of Triplodon corrugatus from five rivers in the north-eastern part of the Amazon region of Brazil








Tajima (D)












Fu (Fs)























Fig. 3

The relationship between genetic distance (Fst) and geographic distance (km) among Triplodon corrugatus populations from the Tapajós, Amazon, Tocantins, Irituia and Piriá in the north-eastern Brazilian Amazon

The plots of ΔK (maximum ΔK = 0.2542) based on the STRUCTURE analyses indicated that the populations sampled in the five Amazonian rivers may be divided into two clusters (K = 2; [Ln Pr(X/K)] = −7289.3), one represented by the Piriá river and a second with the populations from all the other rivers. In the Tocantins river, the haplotypes H4 and H6 displayed a genetic influence from both clusters (Fig. 4).
Fig. 4

Structure diagram showing the genetic contribution of each haplogroup (ΔK  =  2). Each bar represents one individual and colours represent each haplogroup from populations of Triplodon corrugatus from five rivers in the north-eastern Brazilian Amazon

The ML (Fig. 5) and BA (not shown) phylogenetic trees showed that all haplotypes from the Piriá river joined in a single clade (ML bootstrap: 79%, BA frequency: 1.0). The saturation test using COII–COI sequences detected little saturation (Iss = 0.231; Issc = 0.375; P < 0.001).
Fig. 5

Tree topology obtained by maximum likelihood (ML) for Triplodon corrugatus based on COII + COI. Mean ML bootstrap percentages and Bayesian posterior probabilities are shown above and below the branches, respectively


The COII–COI sequences of Triplodon corrugatus from rivers of the north-eastern Brazilian Amazon show moderate to high levels of haplotype diversities, with the exception of the population from the Irituia river, where only a single haplotype was found. The relatively short distance between the Tocantins and Irituia populations, via the Guamá river (Fig. 1), should, in theory, facilitate dispersal via host fishes (Wächtler et al., 2001). This would reduce differences between these populations since gene flow and exchange of genetic material will, sooner or later, tend to homogenize populations (Allendorf & Luikart, 2007). The reason for low diversity in the Irituia is unknown, but may have been the result of a bottleneck or a recent colonization event.

The neutrality tests of Tajima (1989) and Fu (1997) suggested recent population expansion of T. corrugatus in the Tocantins, Tapajós and Amazon rivers. Genetic structure was detected between the Tocantins and Tapajós populations, but not between the Amazon and Tapajós, nor between the Amazon and Tocantins. Such a pattern may be explained by a stepping-stone model of migration, in which migration is greater between populations that are closer to one another than between those that are more distant (Kimura & Weiss, 1964). Isolation by distance may explain heterogeneity between geographically distant populations such as the Tapajós and Tocantins, or the Tapajós and Irituia. However, more sampling effort, especially from western Amazon rivers is needed to further support this in the Amazon as a whole. Additionally, since the present study only analysed maternally inherited mitochondrial DNA, use of nuclear markers could provide evidence for a different history, or support the isolation. Genetic differentiation through isolation by distance occurs in populations of freshwater mussels, for example, in large rivers and their tributaries, such as the Lake Erie and Ohio river drainages (Elderkin et al., 2008) as well as over relatively short distances within a river, such as the Thames (Zieritz et al., 2010).

The hyriid Triplodon corrugatus is only known from the Amazon and Orinoco basins (Pereira et al., 2014). However, this species was found in the Piriá river, which discharges into the Atlantic Ocean, around 500 km east of the mouth of the Amazon, and is not connected to either of the former basins. Therefore, the presence of T. corrugatus in the Piriá river may indicate historical connectivity with the Tocantins basin, perhaps via an extension of the Guamá river towards the Piriá (Fig. 1). All haplotypes found in the Piriá samples were private to this river (Fig. 2). The presence of at least seven mutations between the haplogroup of the Piriá river and that of the Tocantins and Amazon basins, and the high genetic variability in both these haplogroups, lends support for their allopatric evolution. The significant structure found between the Piriá and all the other rivers is maintained by the current absence of connectivity between the Piriá river and the Tocantins and Amazon basins and their tributaries (Fig. 1).

Bottlenecks, founder effects, isolation or admixture of populations may result from geological changes in the drainages of Amazonian rivers (Lundberg et al., 1998; Hoorn et al., 2010). For example, around 10 Mya, a connection was established between the eastern and western rivers of the Amazon region with consequent changes in the direction of the drainage and the position of the mouth of the Amazon river (Lundberg et al., 1998). In addition, more recent neotectonic processes in the Amazonian lowlands have readjusted river drainage patterns (Hoorn et al., 2010; Soares Júnior et al., 2011).

In conclusion, the Triplodon corrugatus population from the Piriá river is diverging from all the other rivers in the eastern and central Amazon region due to geographic isolation, which over generations, if the allopatry persists, may lead to a separate species. The Irituia population has low genetic variability, although it is unclear if this is the result of a bottleneck or a recent colonization event. The populations from the Tocantins and Amazon had the highest genetic diversity, and our data show that these are in demographic expansion.



Guilherme da Cruz Santos-Neto was supported by the Fundação Amazônia Paraense de Amparo a Pesquisa (FAPESPA). This paper was supported by grants from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) – Universal/2006 and from FAPESPA/VALE S.A (Edital 001/2010, Process: 2010/110634; ICAAF 057/2011). We would like to thank João Miranda and his family, Leôncio Braz de Sousa Neto and his family and Ivoneide Ferreira da Silva for providing assistance during fieldwork. Bivalves were collected under licences 22204-1 and 21187-1 from the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio).


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Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Guilherme da Cruz Santos-Neto
    • 1
    • 2
  • Ismael Sander da Silva Nunes
    • 2
  • Colin Robert Beasley
    • 3
  • Adam Rick Bessa Silva
    • 2
  • Cleidson Paiva Gomes
    • 4
  • Claudia Helena Tagliaro
    • 2
    Email author
  1. 1.Instituto Federal de Educação, Ciência e Tecnologia do ParáAbaetetubaBrazil
  2. 2.Laboratório de Conservação e Biologia Evolutiva, Instituto de Estudos CosteirosUniversidade Federal do ParáBragançaBrazil
  3. 3.Laboratório de Conservação da Biodiversidade e das Águas, Instituto de Estudos CosteirosUniversidade Federal do ParáBragançaBrazil
  4. 4.Instituto Federal de Educação, Ciência e TecnologiaBragançaBrazil

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