Analysis of the Mitochondrial D-Loop Reveals That Neither River Boundaries Nor Geographic Distance Structure the Fine-Scale Gene
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Hydrobiologia https://doi.org/10.1007/s10750-019-04069-0 (0123456789().,-volV)( 0123456789().,-volV) PRIMARY RESEARCH PAPER Analysis of the mitochondrial D-Loop reveals that neither river boundaries nor geographic distance structure the fine-scale genetic variation of an Amazonian treefrog Jonatha Edson de Paula Lima . Vladimir Pavan Margarido . Rafaela Maria Moresco . Domingos de Jesus Rodrigues Received: 7 July 2018 / Revised: 30 August 2019 / Accepted: 4 September 2019 Ó Springer Nature Switzerland AG 2019 Abstract While most anurans have limited vagility intrapopulation (81.78%) genetic diversity, with only and local fidelity, there are some exceptions. In the 18.22% of the variation being found among popula- present study, we used Boana boans, a large treefrog tions. Analysis of molecular variance and Bayesian found throughout most of the Amazon basin, as a cluster analysis detected a lack of genetic structuring model organism. We investigated the possible isola- within the study area. The model species presented a tion of the B. boans demes located on opposite capacity for dispersal over long distances in compar- margins of the Juruena River and their population ison with most other amphibians, which, together with structure. We sampled 14 individuals of B. boans and its resistance to desiccation and reproductive mode, analyzed the mitochondrial D-Loop to verify whether enable this treefrog to disperse across rivers and the river or Euclidean distance is acting as barrier to overland. In the specific case of Juruena River, many the dispersal of this frog. The sequencing revealed 12 fluvial islands present within the study area may also haplotypes, with global Fst values of - 0.079, K2P be favorable to the dispersal of the species. values ranging from - 0.187 to 0.054, and primarily Keywords Anura Á Boana boans Á D-Loop Á Landscape genetics Á River barriers Handling editor: Lee B. Kats J. E. de Paula Lima Á D. de Jesus Rodrigues Universidade Federal de Mato Grosso, Po´s-Graduac¸a˜oem Ecologia e Conservac¸a˜o da Biodiversidade, Cuiaba´, Introduction Mato Grosso, Brazil The limited vagility and local fidelity of most J. E. de Paula Lima Á D. de Jesus Rodrigues Universidade Federal de Mato Grosso, Nu´cleo de Estudos amphibians have led researchers to consider anurans em Biodiversidade da Amazoˆnia Mato-grossense – and caudates (less vagile) to be poor dispersers, given NEBAM, Sinop, Mato Grosso, Brazil their morphological and metabolic constraints (Coster et al., 2015; Nowakowski et al., 2015). This designa- V. P. Margarido (&) Á R. M. Moresco Universidade Estadual do Oeste do Parana´, Centro de tion of amphibians as poor dispersers generates a Cieˆncias Biolo´gicas e da Sau´de, Cascavel, Parana´, Brazil degree of inconsistency, especially considering that e-mail: [email protected] many species are widely distributed, in particular in the Neotropics (Reading et al., 1991; Gascon et al., V. P. Margarido Universidade Estadual de Maringa´,Po´s-Graduac¸a˜oem 1998). In fact, anurans may often cover distances of up Biologia Comparada, Maringa´, Parana´, Brazil 123 Hydrobiologia to 10 km (Smith & Green, 2005). At this spatial scale, may help to answer many unanswered or poorly gene flow may be hampered primarily by major resolved questions. barriers such as rivers (Angelone et al., 2011) which Amphibians are considered to be valuable models play a fundamental role in the maintenance of species for investigating the processes that shape the genetic diversity in the tropics, by impeding gene flow structure of populations (Zeisset & Beebee, 2008). In between populations on their opposite margins, and the Amazon region, most of the studies have focused reinforcing possible allopatric speciation (Gascon on dendrobatidis models, and there has been little et al., 1998). work on hylids (Ame´zquita et al., 2009; Kaefer et al., Two theories have been proposed to account for the 2013; Maia et al., 2017). In contrast with these models role of rivers in the zoogeography of vertebrates: (Simo˜es et al., 2014; Maia et al., 2017), Boana boans Wallace’s (1854) river barrier hypothesis and the river (Linnaeus, 1758) is a species of large frog and its refuge hypothesis of Ayres & Clutton-Brock (1992). males have the habit of vocalizing on the banks of the The latter hypothesis assumes that the Amazon Amazonian rivers in their reproductive period; how- rainforest contracted during the glaciations but did ever, these males are also territorialist and philopatric not disappear. This shrinkage reduced forest cover at to the reproductive site (Magnusson et al., 1999). the headwaters of Amazonian rivers, isolating popu- Among the mitochondrial molecular markers is the lations at river mouths. The river barrier hypothesis D-Loop or Control Region (CR), this region evolves has been tested in multiple vertebrate taxa over the much faster than the rest of the mitochondrial gene past 160 years (Wallace, 1854; Gascon et al., 1998; (Brown et al., 1986). This rapid change capacity Bates et al., 2004; Souza et al., 2013; Duarte et al., (changeability) makes the D-Loop segment an appro- 2014). priate marker to address genetic issues at the popula- The displacement and gene flow of terrestrial tion level (Hoelzel et al., 1991), such as population animals are influenced by a series of barriers, from diversity, (Chen et al., 2012; Kawabe et al., 2014), anthropic or natural origin. An example of natural besides (as well as) being useful to test evolutionary barriers is rivers, which exert a barrier function in all relations and biodiversity (Arif & Khan, 2009). taxa (Waits et al., 2015). The Amazonian rivers are Although genetic studies of landscape and diversity able to isolate populations and species of anurans, are essential, there are few studies that use the D-Loop shaping the intraspecific population structure and in anuran amphibians, which would reveal important contributing to the biogeographic regionalization of information about intra- and interpopulation genetic the Anura group (Godinho & Da Silva, 2018; Ortiz diversity (Segelbacher et al., 2010). Genetic diversity et al., 2018). These rivers can act as barriers to among individuals of the same population is an dispersal, where body size of anurans is a determining important factor for fitness to environmental condi- factor in the isolation and distribution of amphibian tions (Takahashi et al., 2018). Therefore, knowledge species (Moraes et al., 2016). of the genetic variability status and its spatial– Studies of river barriers in amphibians based on temporal distribution are fundamental for a correct molecular markers have revealed a number of differ- analysis of the situation and detection of possible ent scenarios, ranging from highly structured popula- threats to a species (Escudero et al., 2003). In the tions forming well-defined clusters (Fouquet et al., present study, we investigated the possible genetic 2012; Kaefer et al., 2013; Maia et al., 2017), to isolation of demes in a local population of B. boans reduced structuring and limited genetic distance through the analysis of molecular diversity and between demes (Gascon et al., 1998; Lougheed population structure. et al., 1999; Funk et al., 2007), and even panmixia (Crawford, 2003; Zeisset & Beebee, 2008). Rivers may play a relevant role in the genetic structuring of Materials and methods many anuran species, although the degree of perme- ability of these barriers depends fundamentally on the Sampling specific characteristics of each species (Fouquet et al., 2015). The application of molecular markers as an We captured the Boana boans specimens in the analytical marker is a relatively new approach that municipality of Cotriguac¸u (09°49009.000 S, 123 Hydrobiologia 58°15031.100 W), in northwestern Mato Grosso, Brazil. auditory searches. While Vitt & Caldwell (2014) The study area encompassed a stretch of approxi- reported the aggregation of choirs of B. boans during mately 6 km of the Juruena River, a third-order the mating season, on the margins of the Juruena tributary of the Amazon, which is 2,700–3,100 m River, individual frogs were separated by distances wide at this point (Fig. 1). The level of the Juruena exceeding 200 m. We collected 14 specimens of B. varies by up to 5 m between the rainy and the dry boans (13 males and one females), four from the seasons. The margins of the river in this area are island, three from the LM, four from RM1, and three covered with well-preserved riparian forest with large from RM2 (the female was captured at the last trees, but few streams. This stretch of the rivers also mentioned location). Specimen collection was autho- has a number of rapids and rocky outcrops, and many rized by SISBIO permanent license 18573-1. We islands, some of which are relatively large, with an extracted a sample of liver tissue from each specimen area of over one hectare. Although we used the sample and preserved it in 100% ethanol for the subsequent size of 14 individuals (from three to five at each point), extraction of the mitochondrial DNA. We fixed all the this is usual among researches with mitochondrial D- specimens and deposited them as vouchers in the loop DNA. For review, see Shaffer & McKnight herpetological sector of the Biological Collection of (1996), Zhong et al. (2008) and Gvozˇdı´k et al. (2010) Southern Amazonia (ABAM: Acervo Biolo´gico da (one or two, three to seven and two to three individuals Amazoˆ nia Meridional) in Sinop, Mato Grosso (Brazil). per population, respectively). Chen et al. (2012) used a sample size of three to nine specimens and considered Extraction of the DNA the variation in his results as somewhat experimental. Tao et al. (2005) sequenced the D-Loop of 28 Andrias We extracted the total DNA using the GenEluteTM davidianus (Blanchard, 1871) salamander to investi- Mammalian Genomic DNA Miniprep kit (Sigma- gate the patterns of genetic structure of four sites.