DNA Barcodes 2015; Volume 3: 110–117

Research Article Open Access

Fábio F. Roxo*, Luz E. Ochoa, Guilherme J. Costa-Silva, Claudio Oliveira Species delimitation in Neoplecostomus (Siluriformes: ) using morphologic and genetic approaches

DOI 10.1515/dna-2015-0014 identified with current . Based on these results Received January 24, 2015; accepted July 9, 2015 we conclude that the molecular approaches are helpful to distinguish species of Neoplecostomus, nevertheless it Abstract: In the present study, we compare the results of is important to combine molecular methodologies with alpha taxonomy (based on morphology), DNA Barcoding current taxonomy in order to identify correctly species method with a 2% genetic divergence threshold, and that recently originated. the GMYC (General Model Yule Coalescent) technique to identify species clusters in Neoplecostomus. We used Keywords: Neotropical , , GMYC partial sequences of mitochondrial COI (Cytochrome model oxidase subunit I) for 59 specimens representing 13 valid species (Neoplecostomus bandeirante, N. jaguari, N. langeanii, N. paranensis, N. yapo, N. botucatu, N. selenae, N. doceensis, N. corumba, N. ribeirensis, 1 Introduction N. microps, N. espiritosantensis and N. franciscoensis) of Neoplecostomus collected in all its distribution area. For In the last decades, advances in the sequencing of the analysis we used Bayesian inference of phylogeny with several molecular markers such as mtDNA fragments relaxed clock methods on an arbitrary timescale, using are combined with morphological methodologies to BEAST. The ultrametrics genes trees obtained for each recognition of the biodiversity [1–3]. The DNA Barcoding tree prior evaluated (Yule, Birth-death and coalescent technique was proposed by Hebert et al. [4] with the Population Size) were used in the GMYC analysis to primary aim to create a standard, unambiguous and identify a time in the tree when the branching rate shifts fast system to identify species, using DNA sequences. (in forward time) from Yule and Birth-Death (species) to a Studies using these methods were already published for coalescent (population) process. We found that the GMYC different groups of organism [3, 5–10], and a significant model using the Yule prior identified 11 groups, closer to percentage of these studies report a high resolution in the the current taxonomy (13 species). GMYC analyses using identification of specimens. Ward [11] noted that closely other ultrametric gene trees estimated under alternative related , with higher genetic divergence (upper than prior such as Birth-Death and Coalescent Population Size, 2%), probably belong to different species. In the same identified 9 groups, results observed in the traditional 2% way April et al. [12] said that species with more than 2% genetic distance threshold, resulting in a low number of of genetic divergence are usually sexual incompatibles. species recognized compared to the number of species However, Barraclough et al. [13] emphasized that the inter- intraspecific threshold is influenced by the substitution rate variation among lineages and from a weak connection *Corresponding author: Fábio F. Roxo, Universidade Estadual to evolutionary theory, putting in doubt the power of this Paulista, UNESP, Departamento de Morfologia, Laboratório de method to identify species. Biologia e Genética de Peixes, Distrito de Rubião Junior s/n, CEP Actually, more complex species delimitation methods 18618-970 Botucatu, São Paulo State, Brazil; E-mail: roxoff@hotmail. have been developed using DNA sequence datasets [e.g. com.br Luz E. Ochoa, Guilherme J. Costa-Silva, Claudio Oliveira, Universi- 14–18], trying to make the species delimitation more dade Estadual Paulista, UNESP, Departamento de Morfologia, Labo- efficient and less subjective. The Generalized Mixed Yule- ratório de Biologia e Genética de Peixes, Distrito de Rubião Junior Coalescent (GMYC) method first introduced by Pons et s/n, CEP 18618-970 Botucatu, São Paulo State, Brazil

© 2015Fábio F. Roxo et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Unauthenticated Download Date | 6/3/16 5:38 PM Species delimitation in Neoplecostomus 111 al. [14] and Fontaneto et al. [19], has become one of the Universidade Estadual Paulista, Botucatu, São Paulo State most popular methods for species delimitation based and (NUP) Coleção Ictiológica do Nupelia, Universidade on single-locus data, and it has been widely used in Estadual de Maringá, Maringá, Paraná State. biodiversity assessments and phylogenetic community ecology. This method considers that the mutations arising 2.2 DNA Extraction and Sequencing in one species cannot spread readily into another species (i.e. independently evolving) [20–21]. Additionally, the Total DNA was extracted from ethanol preserved muscle GMYC method uses an ultrametric tree estimated from samples with the DNeasy Tissue Kit (Qiagen) following the sequences, aims to identify shifts in branching rate of manufacturer’s instructions. Partial sequences of the the tree, from a Yule (species) to coalescent (population) gene cytochrome c oxidase subunit I (COI) was amplified process. GMYC has been used to delimit species in several using polymerase chain reaction (PCR) with the primers poorly known groups of organisms [13, 22–24]. FishF1 (5’-TCA ACC AAC CAC AAA GAC ATT GGC AC-3’) Neoplecostomus Eigenmann & Eigenmann [25] and FishR1 (5’-TAG ACT TCT GGG TGG CCA AAG AAT CA-3’) is a of Loricariidae with species known to be [5]. Amplifications were performed in a total volume of morphologically very similar [26] and to have a recent 12.5 μL with 1.25 μL of 10X buffer (10 mM Tris-HCl+15 mM historical diversification [27–28] with most species arising MgCl2), 0.5 μL dNTPs (200 nM of each), 0.5 μL each 5 mM in the Pliocene and other genera of Neoplecostominae primer, 0.05 μL Platinum® Taq Polymerase (Invitrogen), during Miocene. Morphological characteristics used in 1 μL template DNA (12 ng), and 8.7 μL ddH2O. The PCR species description of Neoplecostomus are based on a series reactions consisted of 30-40 cycles, 30 s at 95°C, 15-30 of small details, mainly the swollen integument around s at 48-58°C (according to species), and 45 s at 72°C. the enlarged odontodes, a ridge over the eyes and a greater All PCR products were first visually identified on a 1% spinelet width. Many of these characters are influenced by agarose gel and then purified using ExoSap-IT® (USB the development stage, as well as, by the sex [3, 26, 29–30]. Corporation) following instructions of the manufacturer. Several species of this genus have been described with the The purified PCR products were sequenced using the “Big help of molecular techniques as allozymes [31–33] or COI DyeTM Terminator v 3.1 Cycle Sequencing Ready Reaction sequences [3]. Thus, the main goal of the present study is Kit” (Applied Biosystems), purified again by ethanol to apply the GMYC model and the standard threshold (2% precipitation and loaded on an automatic sequencer 3130- of genetic divergence) proposed by Ward [11] and April Genetic Analyzer (Applied Biosystems) in the Instituto de et al. [12] to automatically try to identify putative species Biociências, Universidade Estadual Paulista, Botucatu, and compared the results with traditional morphological São Paulo State, Brazil. identification of Neoplecostomus. 2.3 Sequence analysis

2 Material and Methods All individual sequences for each species were initially analyzed using the program Geneious Pro 5.4.2 [34] and a 2.1 Sample collection and preservation consensus sequences were obtained. All sequences were aligned using MUSCLE [35] under default parameters All fishes for this study (Table S1) were collected in and the alignment was inspected by eye for any obvious accordance with Brazilian laws, under a permanent misalignments such as sequencing errors due to scientific collection license in the name of Dr. Claudio contamination, paralogy or pseudogenes. Oliveira. Additionally, our laboratory has special federal Nucleotide variation, substitution patterns, and permission to keep and tissues from a public genetic distances (Table 1) were examined using MEGA 6.0 collection under our care. The species were collected using [36]. To evaluate the occurrence of substitution saturation hand nets, from a variety of locations across southeastern in the sequences, we estimated the index of substitution Brazil. After collection, the animals were anesthetized saturation (Iss) described by Xia et al. [37] and the rate and sacrificed using 1% benzocaine in water; the tissue of transitions/transversions [38] in DAMBE 5.2.31 [39]. sample for each specimen was preserved in 95% ethanol The best-fit nucleotide evolution model for COI gene was for molecular studies, and vouchers were fixed in 10% evaluated using MEGA 6.0 under the information-theoretic formaldehyde for morphological studies. All samples used measure of Akaike Information Criterion with corrections in molecular and morphological analyses were deposited for small sample sizes (AICc). Genetic distance among in (LBP) Laboratório de Biologia e Genética de Peixes, specimens and species were calculated using MEGA 6.0,

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Table 1. Genetic distance and standard deviation calculated with the TN93 model among Neoplecostomus species and specimens of the same species (main diagonal). The bolded numbers indicates the species which have the valued lower than the threshold of 2%. 1 2 3 4 5 6 7 8 9 10 11 12 13 1 – N. bandeirante 0.4% ± 0.2 2 – N. botucatu 4.0% ± 0.1% ± 0.8 0.1 3 – N. corumba 5.0% ± 4.0% ± 0.2% ± 1.0 0.8 0.1 4 – N. doceensis 6.3% ± 5.1% ± 4.9% ± – 1.1 1.0 1.0 5 – N. espiritosantensis 7.8% ± 7.3% ± 6.5% ± 7.7% ± – 1.3 1.3 1.2 1.3 6 – N. franciscoensis 10.1% ± 9.5% ± 9.3% ± 9.6% ± 4.8% ± 1.2% ± 1.6 1.5 1.5 1.5 0.9 0.3 7 – N. jaguari 1.9% ± 2.9% ± 4.2% ± 5.3% ± 7.4% ± 9.0% ± 0.1% ± 0.6 0.7 0.9 1.0 1.3 1.4 0.1 8 – N. langeanii 2.4% ± 2.5% ± 3.9% ± 4.6% ± 7.0% ± 9.2% ± 1.4% ± 0.3% ± 0.6 0.6 0.8 0.9 1.2 1.4 0.5 0.2 9 – N. microps 9.8% ± 9.4% ± 8.9% ± 10.2% ± 5.5% ± 6.2% ± 9.2% ± 9.0% ± 0.7% ± 1.5 1.5 1.4 1.5 1.0 1.1 1.5 1.4 0.2 10 – N. paranensis 2.9% ± 3.0% ± 4.4% ± 5.1% ± 6.3% ± 8.3% ± 2.0% ± 1.5% ± 8.5% ± 0.4% ± 0.7 0.7 0.9 1.0 1.1 1.3 0.6 0.5 1.4 0.2 11 – N. ribeirensis 12.0% ± 11.8% ± 12.6% ± 13.2% ± 12.4% ± 14.2% ± 11.6% ± 10.6% ± 12.4% ± 11.1% ± 0.3% ± 1.8 1.8 1.9 1.9 2.0 2.1 1.8 1.6 1.9 1.7 0.1 12 – N. selenae 4.2% ± 1.0% ± 4.6% ± 5.3% ± 7.6% ± 9.8% ± 3.1% ± 2.7% ± 9.7% ± 3.2% ± 11.8% ± 0.1% ± 0.9 0.4 0.9 1.0 1.3 1.5 0.7 0.7 1.5 0.8 1.8 0.1 13 – N. yapo 6.2% ± 5.4% ± 6.9% ± 7.3% ± 8.8% ± 10.1% ± 5.0% ± 4.7% ± 11.9% ± 4.4% ± 13.3% ± 5.2% ± 1.5% ± 1.1 1.0 1.1 1.2 1.4 1.5 1.0 0.9 1.7 0.8 1.9 0.9 0.4

using the TN93 model with Gamma distribution as the of 10 millions steps were performed and every 1000th best model selected by MEGA 6.0. generation a tree was sampled. Examination of the MCMC samples with Tracer v1.5 [43] indicated convergence and 2.4 GMYC molecular species hypothesis adequate mixing of the Markov chains, with estimated sample sizes beneath the asymptote in the hundreds and The ultrametric gene tree for the GMYC analysis was thousand. After inspection with Tracer, we discarded an estimated using the lognormal relaxed molecular clock appropriate number of steps from each run as burn-in and model, it assumes that the rates of molecular evolution summarized the MCMC samples using the maximum clade are uncorrelated, but log-normally distributed among credibility topology found with TreeAnnotator v1.4.7. [44]. lineages [40] as implemented in program BEAST v.1.6.2 The species delimitation by the Generalized [41] were used for all dating analyses. To investigate Mixed Yule Coalescent model (GMYC) was conducted how the tree priors Yule, Birth Death and Coalescent independently for each ultrametric gene tree for each Constant Population Size would affect the phylogenetic prior with standard parameters (interval = c(0,10)) using estimation and species delimitation using GMYC model a single threshold, while changing the upper and lower for species of Neoplecostomus genus we compared the limit of scaling parameters had no noticeable effect on marginal likelihoods for each model using Bayes factors our results. This analysis was developed in the program estimated in Tracer v1.4. The Bayes factor measures the R version 3.0.0 [45] with the package “splits” (Species strength of the data in changing the prior model odds [42]. Limits by Threshold Statistics) (http://r-forge.r-project. For each prior an ultrametric gene tree was obtained and org/projects/splits). The “gmyc” function in R optimizes each analysis was realized with two independent runs the likelihood function described by Pons et al. [14].

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the same group. Only the 2% threshold of genetic distance 3 Results found N. jaguari plus N. langeanii and N. langeanii plus N. paranensis as member of the same group. Genetic 3.1 Genetic analysis and species delimita- distances among Neoplecostomus species and specimens tion hypothesis of the same species are shown in Table 1. The rate of genetic variation ranges from 1.0±0.4% among N. selenae Sequence data for 589 bp fragment of COI were obtained and N. botucatu, to 14.2 ± 1.9% among N. ribeirensis and N. for 59 Neoplecostomus specimens, the best-fit model franciscoensis. of evolution for the data was TN93 model with Gamma distribution with AICc = 4776.6 The sequences did not show insertions, deletions, stop-codons or sequencing 4 Discussion errors due to contamination or paralogy. The nucleotide In mtDNA data the nucleotide variation are used to frequencies were A = 23.7%, T/U = 25.4%, C = 31.7%, and recognize species [4–6], as well as, the morphological G = 19.2% and the global transition/transversion (Ti/Tv) characters are used in the traditional taxonomy. indicated that the transition ratio was 4.8 times highest Therefore, whether or not the genetic variation form that transversion; likewise Iss.c value was greater than distinct clusters corresponding to species-level entities is the Iss, with R² value of 0.98 and 0.72 for transitions and critical to the proposition of the use of DNA barcoding in transversions respectively, indicating that data are not species identification. Furthermore, other important issue saturated. Bayes factor estimated for tree priors did not related with the delimitation of species boundaries is the show a significant difference (Yule model = -2396.98 ± 0.28; species concept. Several authors have called attention Birth Death model = -2394.14 ± 0.24; Coalescent Constant to situations in which adoption of the biological species Population Size model = -2393.45 ± 0.26). However, we concept leads to the recognition of fewer species taxa than tested and compared how the three different priors (Yule, adoption of one of the alternative species concepts, as well Birth Death and Coalescent Constant Population Size) as, Genotypic cluster, Phenetic or Phylogenetic concepts could affect the species delimitation when applying the [46–48]. Many of the concepts and their associated GMYC model and these results are present in Table 2. Our definitions are incompatible and they can lead to different data represents thirteen morphospecies groups exhibiting conclusions concerning the boundaries and numbers of 86% of the nominal species of Neoplecostomus. Based species [21, 49–50]. Our results show an incongruence on GMYC analysis and among the results obtained using concerning of number of groups of Neoplecostomus different tree priors, GMYC under a Yule prior recognized delimited based on the 2% DNA diverge threshold, the a number of species more related with the number of GMYC model under Yule prior and the morphological morphospecies, identifying 11 groups (Fig. 1). GMYC identification (Table 1; Fig. 1). analyses using an ultrametric tree estimated under a Yule The molecular methods delimited 11 and 9 species prior of branching rates showed that the threshold time based on the application of the GMYC model using Yule found was -0.00520777, indicating the time before which prior and 2% DNA diverge threshold, respectively. The all nodes reflect diversification events and after which all genetic analyses showed that Neoplecostomus selenae plus nodes in the tree reflect coalescent events, the likelihood of N. botucatu, N. bandeirante plus N. jaguari, N. langeanii the null model was 458.4984 and the maximum likelihood plus N. jaguari and N. paranensis plus N. langeanii might of the GMYC model was 459.9386. Using the 2% standard to be the same biological entity (Table 1; Fig. 1), however Barcode threshold of genetic distance we identified 9 morphologically 13 species are recognized. groups (Table 1). Both approaches, the GMYC using Yule The use of GMYC method to species delimitation prior and the 2% threshold, identified N. botucatu plus N. using the Yule prior was able to distinguish more groups selenae and N. bandeirante plus N. jaguari as member of

Table 2. Comparison of group delimitation in Neoplecostomus using different Bayesian processes. Process Number of groups Threshold time Likelihood of Null model Likelihood of GMYC model

Yule 11 -0.00520777 458.4984 459.9386

Birth-Death 9 -0.00707059 464.9980 467.0873

Coalescent Constant Population Size 9 -0.01582767 418.6702 420.8079

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Figure 1. Bayesian consensus tree for Neoplecostomus species obtained from 10 million generations. Numbers after branches are posterior probabilities. Values below 0.95 are not shown. Results from different methods of species delimitation are shown with colored bars at right side of the phylogeny. Red bars represent morphospecies and green bars GMYC method using the Yule model. The upper-left graphic is the Lineage-through-time plot representing the threshold time at -0.00520777 (green line).

Unauthenticated Download Date | 6/3/16 5:38 PM Species delimitation in Neoplecostomus 115 compared with the 2% threshold analysis and came closer with traditional taxonomy to better understand the to the species delimitation of the traditional taxonomy. evolutionary process affecting taxa during the species When we used different priors (Birth-Death or Coalescent delimitation and help in biological conservation. Population Size) the GMYC method delimited 9 groups (Table 2). Similar result was previously reported by Acknowledgments: We wish to thank Renato Devidé Monaghan et al. [22] that suggested that the GMYC model for their help during the collection expeditions. This might be deeply affected by the Markov process chosen research was supported by the Brazilian agencies FAPESP in the Bayesian priors during estimation of the branch (Fundação de Amparo à Pesquisa do Estado de São length. 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