D ( i S a E b P a A R R A H A K B F C M P S 1 F s r ( ( h 0 Fisheries Research 183 (2016) 244–253 Contents lists available at ScienceDirect Fisheries Research j ourna l ho me pa ge: www.elsev ier .com/ locate / f i shres iscordance in the identification of juvenile pink shrimp Farfantepenaeus brasiliensis and F. paulensis: Family Penaeidae): An ntegrative approach using morphology, morphometry and barcoding .S.A. Teodoroa,∗, M. Terossib, F.L. Mantelattob, R.C. Costaa,∗ Laboratório de Biologia de Camarões Marinhos e Dulcícolas (LABCAM), Departamento de Ciências Biológicas, Faculdade de Ciências, Universidade stadual Paulista (UNESP), Av. Eng. Luiz Edmundo Corrijo Coube, 14-01, 17033-360 Bauru, SP, Brazil Laboratório de Bioecologia e Sistemática de Crustáceos (LBSC), Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São aulo (USP), 14040-901, Ribeirão Preto, São Paulo, Brazil r t i c l e i n f o rticle history: eceived 12 October 2015 eceived in revised form 30 May 2016 ccepted 8 June 2016 andled by George A. Rose vailable online 23 June 2016 eywords: razil arfantepenaeus ytochrome oxidase I orphometry enaeoidea pecies discrimination a b s t r a c t It is most difficult to identify the coexistent commercial pink shrimps Farfantepenaeus brasiliensis and Farfantepenaeus paulensis during the juvenile stage, when secondary sexual characteristics are still in the developing period. Differences in width and/or shape of the dorsal furrow on pleonite six has tradi- tionally been the primary character used to discriminate juveniles of these two taxa, but is unreliable. This study aimed to test the hypothesis that the taxonomic morphological characteristics traditionally used to discriminate between these species are not effective for juveniles. Molecular analyses showed that the COI gene is an efficient marker for separating these two species and that traditional character- istics do not allow for the correct identification of juveniles of these species. In addition, we found that only 64% of juveniles identified a priori based on traditional morphological traits and subsequently veri- fied molecularly were identified correctly. After molecular identification of the species, we searched for new morphological traits that could be used for reliable identification of juvenile and adult stages using morphometry and comparative morphology. The carapace length (CL) ranged from 12.5 to 26.9 mm in F. brasiliensis and from 9.4 to 49.5 mm in F. paulensis. We affirmed the efficiency of COI as a molecu- lar marker and identified a new morphological trait that will aid in the discrimination of juveniles of F. brasiliensis and F. paulensis. Contrary to our expectations, the characters identified by morphometric analysis were subtle and difficult to apply in field identification situations. However, when analyzing the external morphology of juveniles, it was possible to identify differences between the species in the anterior margin of gastrofrontal carina in relation to the rostrum teeth. In addition to corroborating the difficulty in identifying these two species, our study confirms the importance of the association between molecular and comparative morphology analyses in a fisheries and biodiversity context. Furthermore, we extended the geographic distribution of Farfantepenaeus subtilis through a new record from southern Cananéia, on the southern coast of São Paulo State. © 2016 Elsevier B.V. All rights reserved. . Introduction The penaeids Farfantepenaeus brasiliensis (Latreille, 1817) and . paulensis (Pérez-Farfante, 1967) are two of the most exploited hrimp stocks off Brazil (Costa et al., 2008). These species rep- esent approximately 18% of the total fished marine crustaceans 57,344.8 t) along the Brazilian coast (IBAMA, 2011). Farfantepe- ∗ Corresponding authors. E-mail addresses: sarahteodoro@gmail.com (S.S.A. Teodoro), mterossi@usp.br M. Terossi), flmantel@usp.br (F.L. Mantelatto), rccosta@fc.unesp.br (R.C. Costa). ttp://dx.doi.org/10.1016/j.fishres.2016.06.009 165-7836/© 2016 Elsevier B.V. All rights reserved. naeus brasiliensis and F. paulensis are both known as “pink shrimp”, and usually no distinction is made between taxa during assess- ments of fishery stocks (Brisson, 1981; Chagas-Soares et al., 1995; IBAMA, 2011). These species are endemic to the western Atlantic coast: F. brasiliensis is distributed from North Carolina (USA) to Rio Grande do Sul (Brazil), and F. paulensis is restricted to an area from Bahia (Brazil) to Buenos Aires (Argentina) (Costa et al., 2003). Considering the high morphological similarity between the two species, identification of adults is based on two specific character- istics: F. brasiliensis has a dorsal furrow on the sixth pleonite that resembles more of an ellipse and gradually increases an then tapers toward the posterior margin (Fig. 1a), and it has a black spot at the dx.doi.org/10.1016/j.fishres.2016.06.009 http://www.sciencedirect.com/science/journal/01657836 http://www.elsevier.com/locate/fishres http://crossmark.crossref.org/dialog/?doi=10.1016/j.fishres.2016.06.009&domain=pdf mailto:sarahteodoro@gmail.com mailto:mterossi@usp.br mailto:flmantel@usp.br mailto:rccosta@fc.unesp.br dx.doi.org/10.1016/j.fishres.2016.06.009 S.S.A. Teodoro et al. / Fisheries Research 183 (2016) 244–253 245 F siliens B j a 1 a a i P t i ( a d t p F s w t t d m ( a a o ig. 1. Differences in the dorsal furrow on the sixth pleonite in Farfantepenaeus bra auru, Brazil). unction of third and fourth pleonites (visible only if it is a fresh dult specimen) (Costa et al., 2003; Pérez-Farfante, 1969, 1970, 971). Males have a petasma with a long distomedial projection nd a curved dorsal region; females have the thelycum with the nterior portion of the sideplates covering the posterior process, .e., they present an unexposed posterior process (Costa et al., 2003; érez-Farfante, 1969, 1970, 1971). By comparison, F. paulensis lacks he spot observed on the pleonites, the dorsolateral sulcus width s narrower and nearly uniform along its entire length (Fig. 1b) Costa et al., 2003; Pérez-Farfante, 1969, 1970, 1971). Males have petasma with a short distomedial projection and a small curved orsal region and females have the thelycum with the anterior por- ion of the sideplates not covering the posterior process, i.e., they resent an exposed posterior process (Costa et al., 2003; Pérez- arfante, 1969, 1970, 1971). It is most difficult to differentiate pink hrimp of F. brasiliensis from F. paulensis during the juvenile stage, hen secondary sexual characteristics are still developing. Thus, he only way to separate species is by observing the dorsal furrow of he sixth pleonite, as described above. In adult males, however, the istal region of the petasma often ruptures during trawling, which akes this character difficult to use for discrimination purposes Costa et al., 2003), and the loss of the black spot on the pleonites fter fixation of F. brasiliensis specimens in alcohol/formalin causes dditional difficulty. In a study performed in the Gulf of Mexico with other species f Farfantepenaeus, molecular techniques indicated that the use of is (a) and F. paulensis (b). Scale bar: 10 mm. Illustrations by Pantaleão, J.A.F (UNESP, “traditional” morphologic characteristics resulted in the misiden- tification of approximately 30% of individuals of Farfantepenaeus duorarum (Burkenroad, 1939) and F. aztecus (Ives, 1891) (Ditty and Bremer, 2011). The authors also emphasized that the characteris- tics used for taxon identification are not always sufficient for the correct differentiation of species, highlighting the importance of the association between molecular analysis and comparative mor- phology in studies concerning diversity patterns, ecological issues and fishery management. Other studies have discussed or illustrated diagnostic features useful for discrimination of adults of F. brasiliensis and F. paulen- sis (see Pérez-Farfante, 1969, 1970, 1971; Costa et al., 2003). In addition, May-Kú et al. (2005) described morphometric differences between early juvenile of F. brasiliensis and F. notialis (Pérez- Farfante, 1967). However, none of these studies jointly evaluated the two pink shrimp species fished along the southern and south- eastern coasts of Brazil, F. brasiliensis and F. paulensis. In this context, our study aimed to test the hypothesis that traditional morphologi- cal characteristics are insufficient to reliably discriminate juveniles of F. brasiliensis and F. paulensis. The main objective was to employ a molecular test to determine whether the traditional morpholog- ical criteria used for a reliable identification of the two species are effective. If the molecular techniques indicated misidentification of individuals of both species, we searched for new morphological traits that could allow for reliable discrimination of juveniles and 2 ies Res a e 2 2 t 2 e t i fi e A t t s s u P t C r i o 1 z t l t m p s t t b g C a t i a 2 g a ( t 2 a o o h ( M a p 46 S.S.A. Teodoro et al. / Fisher dults using morphometry and comparative morphology, consid- ring possible differences between males and females. . Material & Methods .1. Sampling The samples were collected from three main localities off he coast of São Paulo State, Brazil: the northern (Ubatuba; 3◦26′S, 45◦04′W), central (Santos; 23◦57′S, 46◦19′W) and south- rn (Cananéia; 25◦05′S; 47◦55′W) regions (Fig. 2). We selected hese regions to serve as a case study because both species, includ- ng small individuals (juveniles), are frequently targeted during shery activities. Specimens were captured with a commercial fishing boat quipped with trawl nets with mesh sizes of 20 mm and 18 mm. fter each trawling, the biological material was stored on ice and ransported to the laboratory, where each specimen was first iden- ified according to the morphological key (Costa et al., 2003). Each pecimen was individually preserved in 75–90% alcohol and then tored in the Crustacean Collection of the Department of Biology, nder the Faculty of Philosophy, Sciences and Letters at Ribeirão reto, University of São Paulo (CCDB/FFCLRP/USP), Brazil. Addi- ional specimens used in analysis were previously catalogued in CDB collections from Rio Grande do Sul State (Fig. 2). Specimens were first morphologically identified by two esearchers (S.S.A. Teodoro and R.C. Costa) at species level, accord- ng to Costa et al. (2003). Sex identification was performed by bserving the first pair of pleopods (Pérez-Farfante and Kensley, 997; Costa et al., 2003) using a Zeiss® stereomicroscope with a oom from 6.7 x to 45x. Then, molecular techniques were applied o confirm whether the identification was correct. The collection ocation and season were unknown to the identifier a priori, and he outcomes were compared (morphological identification versus olecular identification). Morphometric and comparative mor- hology techniques were applied only to specimens successfully equenced, i.e., individuals with a confirmed molecular identity, hus ensuring a reliable evaluation of the characteristics that are raditionally used for species discrimination. The carapace length (CL) was defined as the linear distance etween corresponding to the orbital angle to the posterior mar- in of the carapace along the dorsal midline. Individuals with a L ≤ 22 mm (smaller individuals on which the morphological char- cteristics traditionally used for identification are more difficult o observe) were classified as “juveniles” (Lopes, 2012), whereas ndividuals (males and females) with CL ≥ 22 mm (diagnostic char- cteristics easier to observe) were classified as “adults”. .2. Molecular analyses Molecular analyses included 103 total specimens (Table 1). The enetic material was obtained and processed in accordance with SISBIO license for sampling and genetic analysis of decapods N # 11777-1, Issue Date: 09/16/2007, issued from CGEN to FLM, he coordinator of the Biota FAPESP Thematic project, Process # 010/50188-8). Considering the wide variability in its evolution- ry rate (Moritz et al., 1987), the mitochondrial gene cytochrome xidase I (COI) was chosen for molecular analysis to examine the ccurrence of inter-population variation within decapod species, as as previously been performed in other studies of shrimp species Carvalho-Batista et al., 2014; Gusmão et al., 2000; Terossi and antelatto, 2012; Vergamini et al., 2011). The COI gene is also used s a “barcode” for most extant animals (Hebert et al., 2003). Tissue extraction, PCR amplification with specific primers, roduct cleanup, and sequencing were conducted following our earch 183 (2016) 244–253 laboratory protocols (Mantelatto et al., 2007, 2009), with specific modifications (Teodoro et al., 2015). Sequence editing was per- formed using the BioEdit program, version 7.0.9 (Hall, 1999). The DNA fragments were aligned using BLAST and the NCBI database assembly (http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi) to confirm their respective identities. Additional sequences from other species were used for comparison: two sequences were retrieved from GenBank (F. californiensis – NC 012738.1 and F. notialis – X84350.2) and two sequences of F. subtilis (CCDB 4717 – KF989462 and CCDB 4676 – KX421864) were generated in this study. The analyses followed methods described by Vergamini et al. (2011) for comparison between species. The sequences were previ- ously aligned with pre-defined parameters in ClustalW (Thompson et al., 1994) and were implemented in BioEdit 7.0.9. Then, a genetic divergence matrix was constructed based on the Kimura 2-parameter model (Kimura, 1980) in MEGA version 5 (Tamura et al., 2011). The number of haplotypes was calculated using DnaSP 4.10.9 (Rozas and Rozas, 1999), and the haplotype networks were constructed using a Median-Joining method in the program Net- work (Bandelt et al., 1999), with data preparation in DnaSP. For the adults, genetic differences in the studied regions were analyzed using Analysis of Molecular Variance (AMOVA) (Excoffier et al., 1992), separately considering the variations at each nucleotide site, in Arlequin 3.1 (Excoffier et al., 2005). After the molecular analysis, voucher specimens that were molecularly identified were catalogued in the CCDB/FFCLRP/USP collection (F. brasiliensis: accession numbers 4477, 4478, 4496, 4532; F. paulensis: 4482, 4483, 4484, 4679), and the sequences were stored in GenBank (F. brasiliensis: KF989360-KF989423; F. paulen- sis: KF989424-KF989461). 2.3. Morphometry All the specimens (adults and juveniles) for which the molec- ular identity was verified were used for morphometric analysis. Although it is most difficult to identify species at the juvenile stage, we aimed to find a characteristic that would be reliable for discrim- ination of both juveniles and adults. Additionally, as morphometric techniques could be applied only in individuals with confirmed molecular identities, the addition of adult specimens to the anal- ysis contributed to a more robust result because morphometric analyses require more individuals than molecular analysis. 2.3.1. linear morphometry A digital caliper (accuracy of 0.01 mm) was used for morpho- metric analysis. For each individual, the following measurements were obtained: CL; carapace height (CH); antennal scale length (ASL); length of the first (PL1), second (PL2), third (PL3), fourth (PL4), fifth (PL5) and sixth (PL6) pleonites; telson length (TEL); uropod endopodite length (UEL); and uropod exopodite length (EXL) (Fig. 3A). These measurements were adapted from those performed by Tzeng (2004), who studied the penaeid Parapenaeop- sis hardwickii (Miers, 1878) and found that these traits are useful for morphometric studies. Regarding plate overlap, measurements were taken exactly as depicted in Fig. 3A, considering the curved abdomen of the species, from anterior to posterior margin of an individual pleon. The allometric equation (y = axb) was used to determine the b value, which is used for calculating the effect of CL variation (independent variable) on the separately measured body structures (dependent variables) (Tzeng, 2004). All of the size characteristics were standardized according to Yi* = Yi [X/Xi]b, where Yi* is the standardized size of the desirable feature, Yi is the size of the fea- ture that will be standardized, X is the mean carapace length of the sample, and Xi is the carapace length of the individual (Tzeng, 2004). http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.ncbi.nlm.nih.gov/blast.cgi S.S.A. Teodoro et al. / Fisheries Research 183 (2016) 244–253 247 Fig. 2. Map showing the four localities of the specimens analysed. The thr Fig. 3. Linear (A) and geometric (B) morphometrics of Farfantepenaeus brasiliensis and F. paulensis. A. Characterization of the measurements taken of each individual of each species: carapace length (CL), carapace height (CH), antennal scale length (ASL), length of the first (PL1), second (PL2), third (PL3), fourth (PL4), fifth (PL5) and sixth (PL6) pleonites, telson length (TEL), uropod endopodite length (UEL) and the uropod exopodite length (EXL). Modified from Pérez-Farfante (1969). B. Landmarks used on the carapace: 1. intersection of anterior margin of carapace and dorsal margin of antennal spine; 2. anterior margin of gastrofrontal carina; 3. gastrofrontal carina region, below and anterior to basal margin of epigastric tooth; 4. dorsoposterior margin of carapace; 5. beginning of the curve of the posterior carapace margin; 6. midway along posterior margin of carapace; 7. posterioventral margin of carapace; 8. midway along ventral margin of carapace; 9. anterioventral margin of carapace in t a J v a l L t A g s i � he pterygostomial region; and 10. intersection of ventral margin of hepatic spine nd hepatic carina. Modified from Chan (2008, p. 852). Illustration by Pantaleão, .A.F (UNESP, Bauru, Brazil). Discriminant analysis (DA) was performed to determine which ariables (body structures) were most informative. Differences mong biological categories and samples were assessed with Wilks’ ambda and the associated F and P statistics (Anastasiadou and eonardos, 2008). The Wilks’ lambda values can range from 0 o 1, where 0 = total discrimination and 1 = non-discrimination. dditionally, a canonical discriminant analysis was performed to raphically show the separation among groups (each sex of both pecies). The significance of canonical variables (Roots) was exam- ned using a Chi-square test. An analysis of covariance (ANCOVA, = 0.05) was performed to compare the slope (b) and linear (a) ee localities sampled from the São Paulo State are showed in detail. coefficients of each morphometric relationship between the stud- ied species. 2.3.2. 2 Geometric morphometry Digital images of the carapace side view were obtained using a Canon 60D camera coupled with a Sigma 17–50 mm f/2.8 lens. The distance from the camera lens to the specimen was standardized and maintained, with adjustments made to focus only. Ten anatom- ical landmarks were defined along the carapace margin (Fig. 3B). All of the landmarks were defined using tpsDig 2:14 software (Rohlf, 2009), following previous standardization criteria (Adams et al., 2004; Zelditch et al., 2004). After digitizing, homologous landmarks were aligned using a generalized least-squares superimposition procedure (Procrustes analysis), and information not related to shape, such as position, orientation and scale was removed (Rohlf and Slice, 1990). The morphological variation was estimated using the thin plate spline (TPS) deformation procedure, which fits an interpolation function to a consensus configuration (Bookstein, 1997), deriving the shape variation in two main components: a uniform component (UC), describing the global variation, and a non-uniform component, describing the local variation in specific regions (Zelditch et al., 2004). Then, an analysis of relative warps (RWs), analogous to a principal component analysis, was performed on the non-uniform components, generating new shape variables (RWs) (Rohlf, 1993). All of these numerical routines were performed using tpsRelw 1.46 software (Rohlf, 2008). More detailed explanations of the morpho- metric methods used here can be found in Rohlf (1990), Adams et al. (2004) and Zelditch et al. (2004). The values of the uniform components and relative warps were compared between species, separately for each sex, using a bal- anced multivariate analysis of variance (MANOVA − Wilks’ test, n = 98) in Statistica 11. Centroid size (CS), defined as the square root of the summed squared distances among all landmarks and the center of gravity (centroid) of the configuration, was compared between species using Student’s t-test. The carapace shape varia- tion along with the relative warps, or uniform axes, was described by deformation grids obtained in the TPS analyses. 2.4. Comparative morphology The purpose of the morphological analysis was to search for new characteristics that had not been identified by the techniques applied previously and/or by the current identification keys. All of the individuals whose molecular identity could be confirmed were thoroughly analyzed based on their morphology using an Olympus 248 S.S.A. Teodoro et al. / Fisheries Research 183 (2016) 244–253 Table 1 Number of individuals morphologically identified before the molecular analysis, by species and by sample region; and number of individuals identified after the molecular analysis. Adults: carapace length >22 mm; Juveniles: carapace length <22 mm. Regions Farfantepenaeus brasiliensis Farfantepenaeus paulensis Adults Juveniles Adults Juveniles Before the molecular analysis Ubatuba 3 8 7 9 Santos 0 6 2 10 Cananéia 3 19 6 26 5♂) S n g 1 3 3 p ( 3 b i t e a t f d a i e r w P g e d 0 t b r g l t ( f w o f 3 b a w j Rio Grande do Sul 0 Total 6 After the molecular analysis All regions 6 (1♀; ZX7 stereomicroscope coupled with a light camera. This search for ew characteristics was guided by studies of other species from this enus (Costa et al., 2003; Ditty and Bremer, 2011; Pérez-Farfante, 967). . Results .1. Molecular analysis From the 103 individuals analyzed, 39 individuals were mor- hologically identified as F. brasiliensis and 64 as F. paulensis Table 1). .1.1. Adults Molecular techniques applied to adult shrimp identified a priori y morphology confirmed that traditional criteria reliably discrim- nate taxa (Table 1). Based on the COI partial gene fragments from he adults of the two species (25 individuals, Table 1) and from oth- rs of the same genus used as outgroups (F. californiensis, F. notialis nd F. subtilis), nine haplotypes were defined (Fig. 4). A separa- ion of the two species occurred, in which adults of F. brasiliensis ormed a separate group from F. paulensis, but haplotypes were not istinct across sampled localities. The analysis of molecular vari- nce (AMOVA) with simple hierarchical structure found that there s no statistically significant difference among the sample sites, ither for F. brasiliensis (AMOVA: within regions = 99.45%, among egions = 0:55%, Fst = 0.005, P = 0.29) or for F. paulensis (AMOVA: ithin regions = 103.03%, among regions = −3.03%, Fst = −0.030, = 0.71). The number of mutations between the two species was much reater than the numbers of mutations among individuals of ach species. These results were also corroborated by a genetic istance analysis: intraspecific genetic distance (F. brasiliensis: –0.6%; F. paulensis: 0–0.4%) was lower than the inter-specific dis- ance (4.3–18.1%). Furthermore, the genetic divergence between F. rasiliensis and F. paulensis was within an interspecific divergence ange (10.1–10.9%), confirming the validity of the two species. This initial analysis was sufficient to demonstrate that 1) the COI ene was effective in separating the two species; 2) the morpho- ogical characteristics used to identify adults were efficient; and 3) here was no genetic structure among regions within each species Fig. 4). Thus, considering the confirmation of such aspects, as the ocus of this study was to verify the identity of juveniles, efforts ere directed toward the followed steps for obtaining sequences f individuals with CL <22 mm, regardless of the localities, in the ollowing analysis. .1.2. Adults + Juveniles In total, 33 juveniles that were morphologically identified as F. rasiliensis and 45 juveniles that were morphologically identified s F. paulensis were sequenced. After the prior analysis with adults, e included juveniles in a new haplotype network (with adults and uveniles together), because there was a clear separation from other 0 4 0 33 19 45 59 (32♀;27♂) 19 (12♀;7♂) 19 (5♀;14♂) species of Farfantepenaeus (used as outgroups) in the adult analysis (Fig. 4). Based on the partial COI gene fragments of the 103 ana- lyzed individuals (25 adults and 78 juveniles), 31 haplotypes were defined, of which 25 represented haplotypes of F. brasiliensis and 6 represented haplotypes of F. paulensis (Fig. 5). This haplotype net- work of juveniles indicated that there is haplotype sharing among individuals morphologically classified into two different species (Fig. 5). In this analysis, we found that only 50 juveniles (approxi- mately 64%) were identified correctly (Fig. 5): 1 specimen that was morphologically identified as F. brasiliensis was actually F. paulen- sis, and 27 specimens that were morphologically identified as F. paulensis were actually F. brasiliensis. Consequently, about 50% of the F. brasiliensis were misidentified as F. paulensis. Additionally, a single specimen of Farfantepenaeus subtilis (Pérez-Farfante, 1967) from Cananéia-SP (CCDB 4676) was identified based on the COI gene. The obtained sequence was compared with other from one specimen collected in Alagoas (CCDB 4717), resulting in an genetic divergence of 0.2%; it was also very different from F. brasiliensis (10.1–10.6%) and F. paulensis (9.2–9.6%). 3.2. Morphometric analyses 3.2.1. Linear Morphometry (Adult + Juveniles) In total, 65 individuals that were molecularly identified as F. brasiliensis (33 females and 32 males) and 38 individuals that were molecularly identified as F. paulensis (17 females and 21 males) (Table 1) were used for morphometric analysis. The CL ranged from 12.5 to 26.9 mm in F. brasiliensis and from 9.4 to 49.5 mm in F. paulensis. Adults were included in the morphometric analysis because the intention was to find a characteristic that was reli- able for the identification of both juvenile and adult specimens. A characteristic that is reliable for juveniles and adults would be very helpful and facilitate identification in field studies. Additionally, because morphometric techniques could be applied only in individ- uals for which the molecular identity was confirmed, the inclusion of adults increased the amount of data analyzed, producing more robust results. The discriminant analysis revealed statistically significant dif- ferences between the two species (p < 0.00) and two statistically significant roots (p < 0.00; root 1, eigenvalue: 168.85, canonical R: 0.99, Wilks’ lambda: 0.00, �2: 761.17; root 2, eigenvalue 8.49, canonical R: 0.95, Wilks’ lambda: 0.05, �2: 275.92). Scores of the canonical variables (Roots 1 and 2) for males and females of both species are presented in Fig. 6. A complete separation of the cat- egories (males and females of each species) with no intersection among groups was formed (Fig. 6). Regarding morphometric relationships between males of F. brasiliensis and F. paulensis, the following variables presented sta- tistically significant differences (ANCOVA, � = 0.05): PL2, PL3, PL5, PL6 and TEL. For females of the two species, there were statistically significant differences in the variables PL1, PL2, PL3, PL5, PL6, TEL, UEL and EXL (Fig. 3a). S.S.A. Teodoro et al. / Fisheries Research 183 (2016) 244–253 249 Fig. 4. Haplotype network according to a median-joining analysis, indicating the distribution of 9 haplotypes of adult Farfantepenaeus spp. The size of the circle of each haplotype is proportionate to its frequency in the sample. The small black circles represent the mean vectors. The white circles represent mutational steps. Fig. 5. Haplotype network according to a median-joining analysis, showing the distribut species (F. brasiliensis and F. paulensis). The size of the circle of each haplotype is proportio White circles represent mutational steps. Fig. 6. Farfantepenaeus brasiliensis and F. paulensis, considering only molecular iden- tification. Graphic generated from the relationship between two variables (canonical discriminants) for the two studied species. ion of 31 haplotypes in 103 individuals (25 adults and 78 juveniles) of the studied nate to its frequency in the sample. Small black circles represent the mean vectors. 3.2.2. Geometric morphometry The geometric morphometry calculations were performed sep- arately for males and females to identify shape differences between the two species. The analysis of the carapace shape of individuals of F. brasiliensis and F. paulensis resulted in 16 relative warps (RW) and 2 uniform components (UCs). For the males, the first four rel- ative warps explained 72.6% of the total morphological variation but were not significantly different among individuals of the two species (MANOVA Wilks’ test: F = 0.308, df = 16; 26; p <0.871). There were also no statistically significant differences between species in uniform components (MANOVA Wilks’ test: F = 0.245, df = 2; 40; p <0.784) or in the centroid size (t = 1.345, p <0.186). Among the females, none of the shape variables differed significantly between species (RW: MANOVA Wilks’ test, F = 1.513, df = 4, 40, p <0.217/UC: MANOVA Wilks’ test, F = 2.574, df = 2; 42; p <0.088). For both males and females, no statistically significant differences were found in the carapace shape between the two species. There was no clear separation between the two species in either sex, and the extreme shapes for each category did not indicate morphological variations that could be used as criteria to discriminate between the two species. The morphological variation found for both sexes was very 250 S.S.A. Teodoro et al. / Fisheries Research 183 (2016) 244–253 F rofron F indivi l l o f 3 a s i i t t o t t n t F ( 4 c b u t b s p F t ( ig. 7. Differences in the position of the vertical line through anterior margin of gast . paulensis, for different carapace sizes, considering only the molecular identity of ength (mm). Illustrations by JAF Pantaleão (UNESP, Bauru, Brazil). arge, as the two main axes explained only slightly more than half f the total variation in the shape (51.1% for males and 53.4% for emales). .3. Comparative morphology Considering that the morphometric analysis (both traditional nd geometric) did not indicate visual structures that could aid in pecies discrimination (separation was only statistical, not visual), ndividuals with a confirmed molecular identity were morpholog- cally compared. In this comparative analysis, differences between he two species could be observed in the anterior margin of gas- rofrontal carina in relation to the rostral teeth (Fig. 7). Considering nly rostral teeth, i.e., excluding the epigastric tooth, a vertical line hrough anterior margin of gastrofrontal carina (AMGC) in Farfan- epenaeus brasiliensis is always aligned with the 3rd rostral tooth, ever anterior to it, and often close to or posterior to the end of he 3th rostral tooth (Fig. 7). In contrast, the end of the AMGC in . paulensis is always anterior to the end of the 2nd rostral tooth Fig. 7). . Discussion We confirmed the initial hypothesis that the morphological haracteristics traditionally used to discriminate juveniles of F. rasiliensis and F. paulensis are not effective, and the techniques sed in our study reinforced this. The molecular results showed hat only approximately 64% of the juveniles identified a priori y morphology were identified correctly. This finding is novel for outhwestern Atlantic penaeid populations. Similar identification roblems and error rates occur with the congeners F. aztecus and . duorarum from the Gulf of Mexico, where molecular identifica- ion indicated that over 30% of individuals had been misidentified Ditty and Bremer, 2011). Additionally, we found that morphome- tal carina (AMGC) in relation to the rostral teeth for Farfantepenaeus brasiliensis and duals. The arrows indicate epigastric tooth, and the values represent the carapace tric techniques did not aid in discrimination of F. brasiliensis and F. paulensis due to plasticity in morphology. Regarding the sampling sites, there was no genetic separation between the two species, a fact supported by the genetic diver- gence, haplotype networks and AMOVA. Such result is reasonable since there are no geographical barriers or habitat disjunction that might serve as a barrier to gene flow among sampled localities. The possible consequences for this homogeneity were recently dis- cussed in Teodoro et al. (2015). The structures identified by linear morphometry that were significantly different among the individuals analyzed here were unfortunately very visually subjective and are thus unreliable for discriminating between F. brasiliensis and F. paulensis. This vari- ability in the morphometric data may also reflect fluctuations in the allocation of energy by individuals (Pérez-Castañeda and Defeo, 2002). Environmental conditions can induce differences in shrimp development rates and phenotype, so that variations dur- ing development can alter morphology (Swain and Foote, 1999; Ditty and Bremer, 2011). In many cases in which it was assumed that the cause of morphometric variance was genetic, culturing individuals of the same group under a variety of experimental con- ditions resulted in different allometric growth patterns (Dumont and D’Incao, 2010). Morphometric variations can be affected by many factors, such as food availability, stress and particular stages of the reproductive cycle (Chu et al., 1995; Pauly, 1984). Dis- crimination of morphologically similar species, especially those of commercial value, is essential, and criteria should be effective and easily applied. Morphological and morphometric characteristics of the rostrum have been widely used to identify penaeid shrimp species, espe- cially juveniles (Dall et al., 1990; Ditty and Bremer, 2011; Heales et al., 1985; May-Kú et al., 2005). Geometric morphometry could only substantiate that morphological variability is too high for any measurement to be of value in discrimination. When observing the external morphology of juveniles of both species, we found differ- S.S.A. Teodoro et al. / Fisheries Research 183 (2016) 244–253 251 Fig. 8. Comparison of the carapace morphology of F. brasiliensis (a) and F. paulensis (b) in relation to the carapace of F. subtilis (c). These species can be discriminated b F. pau F ted by d e ( r f t a b s R j t ( d s i F a h u o i t l i a 2 b y observing the adrostral sulcus; where it ends is narrower in F. subtilis than in arfantepenaeus subtilis also has a longer rostrum than the other two species (indica istal margin of scaphocerite. nces in the position of the AMGC in relation to the rostral teeth Fig. 7). However, we must consider that the position of the AMGC epresents a new discriminatory characteristic that is useful only or the two species analyzed here because other species of Farfan- epenaeus may have similar AMGC positions. The choice of such characteristic is reliable for discrimination, considering that F. rasiliensis and F. paulensis were, until the present study, the only pecies of this genus to occur between the regions of São Paulo and io Grande do Sul. Because a characteristic may not be reliable for uvenile discrimination at the species level when used alone, all of he known characteristics must be used: the sixth pleonite furrow Fig. 1) and the position of the AMGC (Fig. 7). The correct identification of the species is a premise for all bio- iversity knowledge and is essential for basic biology research, uch as ecological investigations of population dynamics studies, mpacts of overfishing and estimates of fishing landings (Pérez- arfante, 1998). In this worrying scenario, over less than 40 years, n 87% reduction in the populations of F. brasiliensis and F. paulensis as been recorded (Neto and Dornelles, 1996); therefore, a better nderstanding of the structure of this stock is urgently needed, not nly due to its high commercial value but also due to the need to mplement more effective management plans, especially regarding he crucial populations of juveniles. The integration of molecu- ar taxonomy and comparative morphology can provide important nformation on diversity patterns and on ecological and evolution- ry principles relevant to fisheries management (Ditty and Bremer, 011). The identification and knowledge of shrimp stocks must e based on more than a single method and should be composed lensis and F. brasiliensis. [Adapted from Pérez-Farfante (1967), Fig. 2a-b, page 88]. an arrow), and the rostral tip extends to or beyond base of flagella, and/or to about of other approaches of stock discrimination, such as life history (growth rate, recruitment, etc.), genetic analysis and laboratory studies, such as larval-stage culture (Begg and Waldman, 1999; Cadrin, 2000; Paramo and Saint-Paul, 2010). 4.1. Extension of the distribution of F. subtilis The known geographic distribution of F. subtilis was extended in this study with a new record from Cananéia (25◦S), on the south- ern coast of São Paulo State. The reported distribution of F. subtilis ranges from Cuba (23–19◦N) along the arc of the Antilles to Cabo Frio (22◦S − Rio de Janeiro, Brazil) (Pérez-Farfante, 1967). Because there are no geographic barriers between the states of São Paulo and Rio de Janeiro, the southern extension of its distribution is not surprising, and we expect other reports in other regions of southern Brazil when more accuracy and caution in species identification are applied. Our finding reinforces the need for more reliable characteris- tics for the discrimination of pink shrimp species. This is why the observation of several morphological criteria is so important: for instance, if we analyze only the sixth pleonite furrow, misidentifi- cation of F. paulensis as F. subtilis may occur. Thus, considering the extremely similar morphology among species, we suggest that the AMGC position must be used to discriminate juveniles only after confirming that the individual is not F. subtilis (Fig. 8). Differences in shape of the carapace dorsal furrow can be used to easily sepa- rate F. subtilis from F. brasiliensis and F. paulensis. Farfantepenaeus subtilis has a longer rostrum than the other two species, and the ros- 2 ies Res t m b ( t t 5 t t n m m i c s t d s F f m o a b A T a p f ( s s 2 F s C d i w t c m p c I R A A B B B B 52 S.S.A. Teodoro et al. / Fisher ral tip extends to or beyond base of flagella, and/or to about distal argin of scaphocerite. There is also a difference in the distance etween termination of adrostral sulcus and carapace termination Fig. 8). Finally, our molecular comparison eliminates doubt about he identification of the specimen collected in Cananéia, confirming he geographical extension. . Conclusion The use of molecular techniques allows for precise species iden- ification; however, due to the high costs associated with these echniques, traditional approaches to species recognition are still eeded. The integration of molecular techniques and comparative orphology, as performed in this study, can provide valuable infor- ation about the diversity of patterns and fishing resources. There s literature available to assist in identification of F. brasiliensis when ompared with certain other species of this genus and reports that how differences between F. paulensis and other species; however, he characteristics indicated by these studies do not aid in the iscrimination between juveniles of F. paulensis and F. brasilien- is. As far as we know, there are no studies comparing juveniles of . paulensis and F. brasiliensis or providing reliable characteristics or discriminating between these two species with highly similar orphologies. Thus, the results of this study confirm the difficulty f identifying these two species and provide morphological char- cteristics that are reliable for use in discriminating between F. rasiliensis and F. paulensis. cknowledgments This paper is part of the multidisciplinary research project emático BIOTA-FAPESP (São Paulo Research Foundation), which ims to produce a fine-scale assessment of the marine deca- od biodiversity of the State of São Paulo. Financial support or this project was provided by research grants from FAPESP Temático Biota 2010/50188-8; grant 2011/16268-7). Additional upport came from Coordenaç ão de Aperfeiç oamento de Pes- oal de Nível Superior − CAPES (Ciências do Mar II Proc. 005/2014 − 23038.004308/2014-14). RCC (305919/2014-8) and LM (304968/2014-5) received research scholarships from Con- elho Nacional de Desenvolvimento Científico e Tecnológico − NPq, and MT is grateful to FAPESP (PD 2011/11901-3). 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