Integrative taxonomy detects cryptic and overlooked fish speciesin a neotropical river basin

Laıs Carvalho Gomes1 • Tiago Casarim Pessali2 • Naiara Guimaraes Sales1 •

Paulo Santos Pompeu3 • Daniel Cardoso Carvalho1

Received: 26 March 2015 / Accepted: 1 July 2015! Springer International Publishing Switzerland 2015

Abstract The great freshwater fish diversity found in theneotropical region makes management and conservation

actions challenging. Due to shortage of taxonomists and

insufficient infrastructure to deal with such great biodi-versity (i.e. taxonomic impediment), proposed remedies to

accelerate species identification and descriptions include

techniques that combine DNA-based identification andconcise morphological description. The building of a DNA

barcode reference database correlating meristic and genetic

data was developed for 75 % of the Mucuri River basin’sfreshwater fish. We obtained a total of 141 DNA barcode

sequences from 37 species belonging to 30 genera, 19

families, and 5 orders. Genetic distances within species,genera, and families were 0.74, 9.5, and 18.86 %, respec-

tively. All species could be clearly identified by the DNA

barcodes. Divergences between meristic morphologicalcharacteristics and DNA barcodes revealed two cryptic

species among the Cyphocharax gilbert and Astyanax gr.

bimaculatus specimens, and helped to identify two over-looked species within the Gymnotus and Astyanax taxa.

Therefore, using a simplified model of neotropical biodi-

versity, we tested the efficiency of an integrative taxonomyapproach for species discovery, identification of cryptic

diversity, and accelerating biodiversity descriptions.

Keywords Integrative taxonomy ! Neotropical !Biodiversity ! Ichthyofauna

Introduction

The precise identification and description of species isessential for biodiversity conservation. Considering the

prediction of approximately 8.7 million species distributed

worldwide, it is estimated that 91 % of aquatic specieshave not been formally described (Mora et al. 2011).

Among the 13,000 freshwater species estimated for the

planet, the described neotropical freshwater fishes com-prise more than 5600 species, but estimates of the predicted

number of species exceed 7000 (Albert and Reis 2011).However, a shortage of taxonomists, insufficient infras-

tructure for successful sampling, the need for taxonomic

revision as well as difficulties accessing information(Agostinho et al. 2005) have delayed advances in biodi-

versity description, leading to what is known as taxonomic

impediment (Taylor 1983). Proposed remedies includeinitiatives of an integrative taxonomy (Dayrat 2005) and

‘‘turbo-taxonomy’’ (Riedel et al. 2013), approaches that

combine DNA-based identification, concise morphologicaldescription, and high-resolution images.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10709-015-9856-z) contains supplementarymaterial, which is available to authorized users.

& Daniel Cardoso Carvalhodanielcarvalho@pucminas.br; carvalho.lgc@gmail.com

1 Programa de Pos-graduacao em Zoologia de Vertebrados,Laboratorio de Genetica da Conservacao, PontifıciaUniversidade Catolica de Minas Gerais, Rua Dom JoseGaspar, 500,Coracao Eucarıstico, Belo Horizonte,MG 30535-901, Brazil

2 Museu de Ciencias Naturais, Laboratorio de Ictiologia,Pontifıcia Universidade Catolica de Minas Gerais, Rua DomJose Gaspar, 290,Coracao Eucarıstico, Belo Horizonte,MG 30535-610, Brazil

3 Laboratorio de Ecologia de peixes, Departamento deBiologia, Universidade Federal de Lavras, Avenida DoutorSylvio Menicucci, 1001, Kennedy, Lavras, MG 37200-000,Brazil

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DOI 10.1007/s10709-015-9856-z

Molecular identification systems through DNA barcodes,

i.e. the use of 650 base pairs (bp) of the mitochondrial geneCOI (cytochrome oxidase subunit I), was proposed by

Hebert et al. (2003) as a standard molecular method for

species identification. The efficiency of DNA barcodeidentification relies on a lower intraspecific COI genetic

distance when compared with interspecific genetic distance

estimations (Hebert et al. 2003, 2004a, b; Waugh 2007). Athreshold of 2 % for species delimitation was proposed after

95 % of 1088 fish barcodes showed intraspecific geneticdistance below 2 % (Ward 2009). However, more complex

algorithms such as RESL, ABGD, CROP, and jMOTU were

subsequently tested for their speed, and effectiveness inrecovering species boundaries, with RESL being selected for

the Barcode Index Number (BIN) system (Ratnasingham

and Hebert 2007). These alternative procedures using DNAbarcoding provide a substitute approach to the traditional

tree-based methods for preliminary biodiversity screening

and species identification.Developments in biodiversity discovery and species

identification can benefit from an integrative taxonomy

method, combining morphological characteristics and astandard DNA identification system such as the DNA

barcoding (Ratnasingham and Hebert 2007). Initiatives

using the methodology of DNA barcoding have been car-ried out to identify the fish fauna in a global context.

Studies using this methodology were conducted in Aus-

tralia (Ward et al. 2005), Canada (Hubert et al. 2008),Africa (Nwani et al. 2011), as well as in Brazilian river

basins such as the Sao Francisco (Carvalho et al. 2011),

Paraıba do Sul (Pereira et al. 2011), and Parana (Pereiraet al. 2013) basins, where the rate of species discrimination

in these studies ranged from 95.0 to 100 %. Moreover,

integrative approaches using DNA barcodes and meristicanalysis may be useful, not only to flag potential over-

looked species or operational taxonomic units (OTUs), but

also to indicate potential new species (i.e. candidate spe-cies). Therefore, pipelines for delineation of putative spe-

cies have been proposed as an efficient start for taxonomic

work flows (Kekkonen and Hebert 2014).The Mucuri River basin is located in a set of indepen-

dent basins that drain the eastern region of Brazil in the

northeastern Atlantic Forest freshwater ecoregion, whichpossesses a high level of fish endemism and great bio-

geographic significance. Its ichthyofauna is composed of

61 fish, including 12 marine species, distributed in 45genera and 26 families (Pompeu 2010), and seven non-

indigenous species (Leporinus macrocephalus, Prochilo-

dus costatus, Clarias gariepinus, Cichla sp., Oreochromisniloticus, Cyprinus carpio, and Poecilia reticulata).

Impacts on fish populations and fishing activities in the

Mucuri River have been reported due to intense anthro-pogenic changes, reflected in the high number of

introduced species, as well as deforestation, dumping of

domestic and industrial sewage, and damming for hydro-power generation (Pompeu 2010).

Here we conducted a comprehensive inventory and

developed a barcode library integrated with a detailedmorphological identification of 75 % of the freshwater

ichthyofauna from the Mucuri River basin. The low species

richness of the Mucuri ichthyofauna provides a simplifiedmodel of neotropical biodiversity, enabling the ability to

test the efficiency of an integrative taxonomy approach forspecies discovery, identification of cryptic diversity, and

accelerating biodiversity descriptions.

Materials and methods

Study area

The Mucuri River basin (MRB) is part of a set of inde-pendent basins that drain the eastern region of Brazil, and it

possesses a total drainage area of 15,413 km2 (MMA

2006). It is bordered in the northwest by the JequitinhonhaRiver basin and in the south by the Sao Mateus and Itaunas

River basins (MMA 2006). The temperature of the MRB

waters range from 23 to 27 "C; the climate is consideredsemi-humid and has a dry period lasting 4–5 months per

year (IGAM 2008).

Sampling

Two campaigns for fish collection were held in 2013 and2014, which included 14 sampling sites along the upper

and middle MRB (Fig. 1). Specimens were captured using

gill nets, cast nets, and sieves. Fin fragments were obtainedfrom freshly captured fish and fixed in 100 % ethanol.

Subsequently, samples were taken to the laboratory and

kept at -20 "C. Voucher specimens were fixed in 10 %formalin and later preserved in 70 % ethanol and are held

in the Ichthyology Collection of the PUC Minas Natural

History Museum.All specimens were photographed, geo-referenced, and

identified to the lowest taxonomic level using meristic-

based identification keys and taxonomic papers (Vari 1992;Albert et al. 1999; Castro and Vari 2004; Zanata and

Camelier 2009) (Supplemental Materials).

DNA barcoding

Whenever possible, a minimum of five specimens perspecies were analyzed. DNA extraction was achieved by

salting out following an adapted protocol from Aljanabi

and Martinez (1997). Partial sequences of the cytochromeoxidase I gene (COI) were amplified by polymerase chain

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reaction (PCR) using primers described by Ward et al.

(2005). The PCR contained a final volume of 10 ll,including 1.0 ll 109 buffer containing MgCl2; 0.3 ll ofdNTP (10 mM); 0.25 ll of each the two primers (10 lM);

0.2 ll of Taq DNA polymerase (5 U/ll); 7.0 ll of ultrapure water and 1.0 ll of DNA template. Amplifications

were performed using a thermocycler (Veriti# 96—Well

Thermal Cycler, Applied Biosystems), and they consistedof an initial denaturation step of 2 min at 95 "C followed

by 35 cycles of denaturation for 30 s at 94 "C; 54 "C for30 s for primer annealing, 1 min at 72 "C followed by

10 min at 72 "C. The amplified DNA products were visu-

alized on 1 % agarose gel, and positive amplifications wereselected for DNA sequencing.

Sequencing reactions were performed bi-directionally

according to the manufacturer’s instructions using the BigDye# Terminator Cycle Sequencing Kit v3.1. The

sequencing reaction had a final volume of 10.0 ll, con-taining 2.0 ll of 59 Buffer, 0.5 ll of Big Dye, 1.0 ll ofprimer (10 lM), 5.5 ll of ultrapure water, and 1.0 ll of theamplified DNA product. DNA sequencing was conducted

in an automated DNA analyzer ABI 3500 (LifeTechnologies).

Data analysis

DNA sequences were edited using the software DNA

Baser# v.3.5.4 and aligned using the MUSCLE alignmenttool (Edgar 2004). The intra and interspecific genetic dis-

tances and nearest neighbor distance (NND) were obtained

from the BOLD database platform (Ratnasingham andHebert 2007). Genetic distances were estimated using the

Kimura-2-parameter (K2P) nucleotide evolution model

(Kimura 1980). The Mega 5.1 program was used to con-struct neighbor joining (NJ) (Saitou and Nei 1987) den-

drograms and to estimate the K2P genetic divergence

between clades.All data including the electropherograms, fish photos,

GPS coordinates of each sample site, vouchers numbers,

and detailed taxonomic identifications were deposited inThe Barcode of Life Database platform (BOLD) under the

Barcoding of Neotropical Fish Species Project from theMRB. On-line bioinformatics tools implemented in the

BOLD platform were used to estimate intra and inter

genetic distances between species, genera, and families.

Delimitation of candidate species

A diverse range of statistical methods was used for species

delimitation. First, traditional indexes indices such as

nearest neighbor distance (NND) were used to estimate theminimum genetic distance between pairs of species, and

the barcode gap was calculated on BOLD Workbench

(http://www.boldsystems.org). Second, two clusteringmethods, namely the Barcode Index Number System (BIN;

Ratnasingham and Hebert 2013) and Automatic Barcode

Gap Discovery (ABGD; Puillandre et al. 2012), wereapplied using the barcode threshold of approximately 2 %

as suggested by Ward (2009) who analyzed barcode

sequences of 1088 fish species. ABGD and BIN applyclustering algorithms to distinguish partitions in the genetic

distances among a group of individuals, using a two-phased

procedure to sort sequences into hypothetical species.ABGD first uses a range of prior intraspecific divergences

Fig. 1 Map of the Mucuri River basin depicting sampling sites (red dots). The two dark blue dots represent two hydroelectric dams, Mucuri inthe upper and Santa Clara in the lower portion of the river. (Color figure online)

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to divide the data into groups based on a statistically

inferred barcode gap, and then recursively applies the sameprocedure to the groups obtained in the first step. Whereas,

the approach of BIN firstly employs single linkage clus-

tering coupled with a 2.2 % threshold to establish prelim-inary species boundaries followed by Markov clustering.

This biphasic process has the goal of both improving and, if

needed, redefining groups recovered in the first phase ofboth methods. ABGD analyses were performed using the

web interface (http://wwwabi.snv.jussieu.fr/public/abgd/,web version ‘October 28 2014’) with a relative gap width

value of X = 1.0 and two available distance metrics [JC69

(Jukes and Cantor 1969) and K2P (Kimura 1980)], whilethe other parameter values employed defaults. Assignments

for intraspecific divergence (P) values between 0.001 and

0.100 were recorded. The Barcode Index Numbers (BIN)was automatically estimated in BOLD Workbench.

We followed the approach presented in Padial et al.

(2010) and the congruence among BIN, ABGD and geneticdistance methods to designate candidate species. When

referring to a particular candidate species, we used the

genus or a combination of the binomial species name of themost similar or closely related nominal species followed

(in square brackets) by ‘‘Ca’’ (for candidate) plus a con-

cordant attached Barcode Index Number (BIN), where thenumber refers to a highly divergent barcode sequence of

the species.

Results

We obtained 141 sequences of the COI gene for 37 species,

representing 75 % of the described freshwater fish fauna

from the Mucurı River basin, a large neotropical freshwaterbasin heavily impacted by anthropogenic activities. DNA

barcodes had an average of 658 bp, and no insertions,

deletions, and stop codons were observed. A mean of 3.81specimens were analyzed per species, belonging to 30

genera, 19 families, and 5 orders: Characiformes (38 %),

Siluriformes (38 %), Perciformes (14 %), Cyprinodontif-ormes (5 %), and Gymnotiformes (5 %). Only one speci-

men per species was obtained for Gymnotus sylvius,

Hoplosternum littorale, Imparfinis minutus, Pachyurusadspersus, and P. costatus, and therefore, they were not

included in the estimation of intraspecific divergences. The

NJ tree encompassing all analyzed fish species showedspecies-specific clades and an absence of shared haplotypes

among species (Fig. 2). The estimated K2P genetic dis-

tances within species, genera, and families were 0.74, 9.5,and 18.86 %, respectively (Table 1). The mean intraspecific

value observed was 0.74 %, ranging from 0 to 3.24 %. Of

the 37 species analyzed, 28 had a mean intraspecific geneticdivergence smaller than 1 %.

The nearest neighbor distance analysis (NND) showed

that all species had a K2P divergence higher than 2 % totheir closest neighbor. When considering the 2 %

intraspecific genetic distance threshold for species delimi-

tation, 35 (95 %) fish species were correctly identified.Only two species Cyphocharax gilbert and Astyanax gr.

bimaculatus had intraspecific genetic divergences higher

than 2 %, with mean values of 1.61 % (ranging from 0 to3.24 %) and 0.92 % (ranging from 0 to 2.9 %), respec-

tively. Reflecting the deep intraspecific divergent lineages,the NJ tree clustered C. gilbert and A. gr. bimaculatus

specimens in two distinct clades (Fig. 2).

The BIN analysis identified 39 clusters, including 22taxonomically discordant, 16 concordant, and one single-

ton (Pachyurus adspersus). Cyphocharax gilbert and

Astyanax gr. bimaculatus were separated in two distinctBINs (Fig. 2).

ABGD analysis detected 61–33 candidate species when

varying the prior maximal distance from P = 0.001 toP = 0.1000, respectively, using both the K2P and JC69

nucleotide evolution methods. We chose the partition that

recovered 38 groups (intraspecific distance P = 0.0129and P = 0.0215), since this partition was more consistent

with our a priori morphological taxonomic analysis. ABGD

species delimitation was in agreement with all the BINclusters with one exception; Astyanax gr. bimaculatus was

considered as a single cluster by ABGD (Fig. 2).

Cyphocharax gilbert specimens (n = 6) showed a meanintraspecific value of 1.61 % and were grouped in two

clades in the NJ tree, with an average K2P genetic diver-

gence between clades of 2.9 %, ranging from 2.8 to3.24 %. Within the clades, the divergences ranged between

0.3 and 0.08 % (Fig. 2). Exclusive meristic morphological

characteristics frequently used for Curimatidae speciesidentification could not be found for each candidate species

(Table 2). Moreover, the BIN analysis recovered 2 distinct

BINs, one grouping C. gilbert specimens from MRB andthe neighboring Doce River basin (ACK1539) and the

other with fish exclusive to the MRB (ACN3641).

Astyanax gr. bimaculatus specimens (n = 8) had a meanintraspecific value of 0.92 % and were split in two major

clades (Fig. 2) with an average K2P genetic divergence

between clades of 2.7 %, ranging from 2.5 to 2.9 %. Oneclade included only one specimen identified as Astyanax gr.

bimaculatus (BIN ABY8634) and despite showing a

divergence of 2.7 % when compared to other individuals(n = 7) of the same species, no morphological differences

were found. All specimens presented a horizontally oval

black humeral spot, a lozenge-shaped caudal-pedunclespot, continuing to the tip of the middle caudal-fin rays,

similar to the meristic morphological characteristics

described for Astyanax identification and presented inGarutti (1998). Therefore, no significant morphological

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divergence was registered for the two recovered BINs(ABZ1711 and ABY8634).

One specimen of Astyanax sp. (BIN ACJ1244) had a

genetic divergence of 18.5 % to other congeneric individ-uals and a similarity of 99.8 % with Astyanax vermilion

previously deposited in the BOLD database. The morpho-

logical identification of Astyanax sp. was divergent fromthe A. vermilion description presented in Zanata and

Camelier (2009). Astyanax vermilion possessed a dark

distal portion of the distal pelvic fin, 32–34 lateral linescales, 20–23 branched anal-fin rays, five series of scales

between dorsal-fin origin and lateral line and no more than

five cusps on the dentary teeth. The undescribed species

Astyanax sp. presented homogeneous pelvic fin color, 35lateral line scales, 21 branched anal-fin rays, six series of

scales between dorsal-fin origin and lateral line and seven

cusps on dentary teeth.Incongruences between morphological and barcode

identifications were observed within the Prochilodus and

Gymnotus species. One specimen identified as P. costatusaccording to the meristic characteristics presented in Castro

and Vari (2004) had a genetic similarity of 100 % with the

species Prochilodus argenteus (BOLD number BSB392-10) previously deposited in BOLD. Gymnotus carapo had

an average intraspecific genetic distance of 2.42 %

(0–5.05 %), but it was possible to correctly identify them

Fig. 2 NJ tree based on K2P distances, BIN, and ABGD of all species analyzed. Photos and detailed NJ tree of species with intraspecific K2Pgenetic distances greater than 2 % are shown: a Astyanax spp.; b Cyphocharax gilbert; and c Gymnotus sylvius/G. carapo

Table 1 Genetic distances(K2P) between species, genera,and families

Minimum (%) Mean (%) Maximum (%) Standard Error (%)

Within species 0 0.74 3.24 0

Within genus 4.29 9.5 18.44 0.07

Within family 3.04 18.86 26.02 0.01

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as G. sylvius and Gymnotus aff. carapo based on a mor-

phogenetic approach. Gymnotus sylvius (BIN AAB6215)exhibited wider white interbands than dark band regions,

and the dark bands were divided into blotches and spots

above the lateral line (Albert et al. 1999), which were notobserved for the other specimens identified as Gymnotus

aff. carapo (BIN AAB6216) (Fig. 2).

Discussion

Herein, we implemented an integrative approach that

aimed to test the pipeline accuracy in the identification ofthe freshwater fish fauna from the MRB. Barcode analysis

recovered 38–39 putative species from the 37 morpholog-

ically identified species analyzed, which represents around75 % of freshwater ichthyofauna of the MRB.

The mean K2P genetic intraspecific divergence was

0.74 %, 13 times smaller when comparing to the meaninterspecific distance (9.5 %). Therefore, a barcode gap

was successfully recovered, since no overlap of intra and

interspecific divergence was observed. Neotropical fresh-water fish barcode surveys have reported similar mean intra

and interspecific genetic distance values, ranging from 0.50

to 10.61 % for the Sao Francisco River (Carvalho et al.2011), 0.13 to 10.36 % for the Paraıba do Sul River (Per-

eira et al. 2011), and 1.3 to 7.1 % for the Upper Parana

River basin (Pereira et al. 2013).By applying the barcode threshold of 2 % defined by

Ward (2009), we were able to differentiate all analyzed

species from the MRB. The NND reinforced this result byshowing that all species had a higher than 2 % K2P

divergence to their closest neighbor.

Overlooked cryptic species were flagged within the C.gilbert specimens due to the intraspecific divergence above

2 %, and corroborated by distinct BIN and ABGD clusters

(Fig. 2b). However, only one species of Cyphocharax isreported for the MRB (Vari 1992). The absence of mor-

phological differences between distinct clades, supported

by both BIN and ABGD analysis, clearly indicates a casefor a new candidate cryptic species. Moreover, since one

BIN was shared with a Cyphocharax species from the

neighboring Doce River while the other one was exclusive

to the MRB, we suggest the occurrence of a new candidatespecies, namely Cyphocharax aff. gilbert [ACN3641],

endemic to the MRB. With the growth of the barcode

database, including all other neighboring river basins, wemay be able to expose endemic cryptic species using BIN

analysis as well as decreasing the risk of synonymies.

Astyanax gr. bimaculatus had a 2.7 % mean intraspecificdivergence between NJ tree clades and recovered BINs

(Fig. 2a) but with no ABGD or morphological support. Thegenus Astyanax is a well know complex of species and one

of the most specious neotropical fish groups with over 140

described species (Froese and Paulay 2010; Eschmeyer2015), occurring in practically all river basins of the

neotropics (Marinho and Lima 2009). Eigenmann (1921)

redescribed Astyanax bimaculatus as being comprised ofseveral subspecies, and Garutti (1995), reviewing nominal

species, recognized 30 valid species, suggested the need for

more systematic studies to objectively determine the actuallevels of diversity within the bimaculatus complex.

Interestingly, a deep divergence of 18.5 % was found

when comparing one unidentified specimen of Astyanax sp.(ACJ1244) to other congeneric specimens sampled from

the Mucuri basin. This highly divergent haplotype had high

similarity (100 %) to an A. vermilion barcode sequencepreviously deposited in the BOLD database. However, A.

vermilion is not described for the Mucuri basin, but from a

coastal northeastern Brazilian river 300 km distant, andmorphological analysis did not corroborate the barcode

identification. Morphologic variation wider than described

for A. vermilion or a mistake in the morphological identi-fication of the species deposited as A. vermilion in BOLD

may explain this discordance.

The native species Prochilodus vimboides could beclearly differentiated from the introduced species P.

costatus by DNA barcoding, since interspecific divergence

of 9 % was recovered. Nonetheless, the non-indigenousspecies identified morphologically as P. costatus had a

barcode similarity of 100 % with another non-indigenous

species P. argenteus (BOLD number: BSB392-10). Thedivergence between the morphological and molecular

identification of P. costatus may indicate the presence of

Table 2 Morphological identification of Cyphocharax gilbert specimens based on diagnostic characteristics

Specimen BIN Number oflateral line scales

Number of scales abovelateral line to origin of dorsal fin

Number of scales belowlateral line to origin of anal fin

LGC 4091 ACK1539 37 6! 5!Mu014 ACK1539 39 7! 5!LGC 4112 ACN3641 38 7! 5!LGC 4105 ACN3641 38 6! 5!LGC 4114 ACN3641 37 7! 5!LGC 4113 ACN3641 39 6! 5!

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Prochilodus hybrids in the Mucuri basin between the two

species of the Sao Francisco River. Hybrids of Prochilodusare commonly produced in aquaculture farms in Brazil and

may escape into the wild.

In the case of the electric knifefishes, the integrativeapproach based on molecular taxonomy and morphological

characteristics allowed the precise identification of the

overlooked species G. sylvius, not yet reported for theMRB. All Gymnotus specimens were first identified as G.

aff. carapo and due to their high intraspecific geneticdivergence and the presence of two clades in the NJ tree

(Fig. 2c), the specimens were subjected to a more detailed

morphological analysis, allowing their correct taxonomicidentification. However, G. sylvius is described only for the

Ribeira de Iguape, Paraıba do Sul, and Upper Parana River

basins, but not for the MRB (Albert et al. 1999; Pompeu2010). Therefore, its distribution range should be expanded

to the MRB based on the results of our integrative

approach.This integrative approach using a comprehensive

inventory through the development of a barcode library

combined with a detailed morphological identification wasa robust methodology for species delimitation. For

instance, our results indicated a new candidate species

(Cyphocharax aff. gilbert [Ca ACN3641]) and detectedoverlooked species within the Astyanax (A. vermilion) and

G. sylvius specimens. Therefore, our integrative pipeline

using a simplified biodiversity model (i.e. Mucuri ichthy-ofauna) showed a possible remedy to the taxonomic

impediment, and may be used to adequately describe and

conserve the biodiversity of other complex neotropicalichthyofaunas.

Acknowledgments We thank Angelo Monteiro, Danilo Neto, IagoPenido and Ivo for helping with the fieldwork and laboratory analysis.DCC is grateful to Conselho Nacional de Desenvolvimento Cientıficoe Tecnologico (CNPq) for his productivity fellowship (308537/2014-9). This work was funded by the Brazilian Barcode of Life NetworkBrBol/CNPq (564953/2010-5), Coordenacao de Aperfeicoamento dePessoal do Ensino Superior (CAPES—PRO EQUIPAMENTOS GrantNumber 783380/2013), Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico—CNPq (Process Numbers INCT573899/2008-8 and 482852/2011-9) and Fundacao de Amparo aPesquisa do Estado de Minas Gerais—FAPEMIG (Process NumberINCT APQ-0084/08).

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