High genetic connectivity among pink shrimp Farfantepenaeus paulensis (Pérez-Farfante, 1967) groups along the south-southeastern coast of Brazil

Journal Pre-proof High genetic connectivity among pink shrimp Farfantepenaeus paulensis (PérezFarfante, 1967) groups along the south-southeastern coast of Brazil S.S.A. Teodoro, M.C. da Silva Cortinhas, M.C. Proietti, R.C. Costa, L.F.C. Dumont PII:

S0272-7714(19)30436-6

DOI:

https://doi.org/10.1016/j.ecss.2019.106488

Reference:

YECSS 106488

To appear in:

Estuarine, Coastal and Shelf Science

Received Date: 3 May 2019 Revised Date:

13 October 2019

Accepted Date: 18 November 2019

Please cite this article as: Teodoro, S.S.A., da Silva Cortinhas, M.C., Proietti, M.C., Costa, R.C., Dumont, L.F.C., High genetic connectivity among pink shrimp Farfantepenaeus paulensis (PérezFarfante, 1967) groups along the south-southeastern coast of Brazil, Estuarine, Coastal and Shelf Science (2019), doi: https://doi.org/10.1016/j.ecss.2019.106488. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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1

High genetic connectivity among pink shrimp Farfantepenaeus paulensis (Pérez-

2

Farfante, 1967) groups along the south-southeastern coast of Brazil

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*Teodoro, S.S.A.

1,2,3

; da Silva Cortinhas, M. C.

2, 3

2

1

; Proietti, M. C. ; Costa, R.C. ; Dumont, L. F. C.

3

1

Laboratory of Biology of Marine and Freshwater Shrimp (LABCAM), University of State of São Paulo (UNESP), Av. Eng. Luiz Edmundo Corrijo Coube, 14-01, CEP 17033-360, Bauru, SP, Brazil 2 Laboratory of Marine Molecular Ecology (LEMM), Rio Grande Federal University (FURG), Campus Carreiros, Av Itália, km 8, Campus Carreiros, CEP: 96201-900, Rio Grande, RS, Brazil 3 Laboratory of Decapod Crustaceans (LCD), Rio Grande Federal University (FURG), Campus Carreiros, Av Itália, km 8, Campus Carreiros, CEP: 96201-900, Rio Grande, RS, Brazil *Corresponding author: [email protected]

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ABSTRACT The pink shrimp Farfantepenaeus paulensis is one of the most important fishing resources along the south-southeast coast of Brazil. Its fishing zone includes two main reproductive stocks, located at the coasts of Santa Catarina and São Paulo states. The species life cycle includes an offshore reproductive stage and juvenile recruitment to estuarine areas; therefore, adults are caught in oceanic areas by industrial fisheries and juveniles in estuarine areas by artisanal fisheries. The unrestricted growth of the industrial fleet, along with the increase of artisanal fishing in estuarine areas and the low effectiveness of fishery legislation, led to a collapse of the pink shrimp fishery in the late 1990s. The present work aimed to assess the population genetics of the pink shrimp F. paulensis throughout its main distribution in the Southwestern Atlantic (SWA), to verify whether different fishing stocks also represent different genetic stocks. Based on the evaluation of the mtDNA control region (D-loop), we detected high connectivity and absence of genetic structure among F. paulensis groups in the SWA. Fixation indexes, Analysis of Molecular Variance and a haplotype network also showed no significant genetic differences, with individuals sharing haplotypes among more than one region. Mismatch analysis showed unimodal distributions for all the sampled populations with high haplotype diversity and low nucleotide diversity, showing a pattern of recent demographic expansion, which was corroborated by the Tajima's D and Fu's Fs neutrality tests. Estimates of migration rates indicated high gene flow, with a large number of migrants exchanged between regions. All obtained results are consistent with a panmitic population of F. paulensis. This scenario is a result of a complex interaction of several factors, from the influence of currents on larval dispersal to the effect of overfishing. The knowledge on the diversity and genetic structure of F. paulensis is a fundamental part of the scientific basis necessary for decision makers to elaborate adequate management strategies for pink shrimp stocks.

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Keywords: mtDNA Control Region (D-loop); genetic diversity; population connectivity; demographic analysis; genetic monitoring.

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1. INTRODUCTION

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Shrimp catch in South and Southeast Brazil moves several sectors of the fishing

48

industry, and high commercial values compensate the relatively limited production

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(PEZZUTO, 2001). Among the shrimp species fished along the Brazilian coast, the pink

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shrimp Farfantepenaeus paulensis (Pérez-Farfante, 1967) is one of the most exploited

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fishery resources in the S/SE regions (IBAMA, 2011). This species is distributed in the

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Southwestern Atlantic (SWA) from Bahia state (14°47’S, 39°02’W; Brazil) to the coastal

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waters of Buenos Aires (34°36’S, 58°22’W; Argentina), and is easily found at depths up

54

to 80m (D’INCAO, 1991; COSTA et al., 2003).

55

Farfantepenaeus paulensis spawns in oceanic waters, and planktonic stages

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migrate towards estuaries until the end of larval development; after four to ten months,

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juveniles leave the estuary towards the oceanic zone for reproduction, completing their

58

lifecycle (D’INCAO, 1991; COSTA et al. 2008). The pink shrimp fishery exploits the two

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population strata, with juveniles and pre-adults caught by artisanal fishing in estuarine

60

areas, and adults caught by industrial fishing in oceanic areas (D’INCAO et al., 2002).

61

The uncontrolled growth of the industrial fleet, together with the increase in artisanal

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fishing in estuaries and the low effectiveness of fisheries legislation, have led to a

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collapse of the industrial shrimp fishery in the late 90’s (D'INCAO et al., 2002;

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HAIMOVICI & CARDOSO, 2017). The industrial fleet has therefore shifted from mono

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to multispecific, exploring other shrimp species to be economically sustainable

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(D'INCAO et al., 2002). The offseason period changed and increased its coverage in

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1996 – becoming mandatory along the coast of Espírito Santo to Rio Grande do Sul

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states – and, since 2002, includes not only pink shrimp but also all other exploited

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penaeids (FRANCO et al., 2009). However, considering that official fishing statistics

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are obtained without the separation of pink shrimp species (F. paulensis, F. brasiliensis

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(Latreille, 1817) and F. subtilis (Pérez-Farfante, 1967)) there is no reliable estimate of

3

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the total amount of F. paulensis fished in Brazil. Such imprecision hampers

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assessments of the degree in which overexploitation threatens F. paulensis. For

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instance, in the Brazilian Red List of Crustaceans (PINHEIRO & BOOS, 2016), F.

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paulensis is categorized as “insufficient data" (BOOS et al., 2016); however, a recent

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study revealed a decrease in the maximum sizes of F. paulensis as indicator of

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overfishing (NOLETO-FILHO et al., 2017).

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Establishing limits for the exploitation of fishery resources requires reliable

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information on the gene flow and number of migrants exchanged between different

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regions, as populations affected by anthropogenic or natural pressures may or may not

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be re-established by individuals from neighboring areas (KORDOS et al., 1993;

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LACERDA et al., 2016). Defining the limits of a stock and its power to export or import

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biomass (i.e. individuals) is essential for obtaining reliable population parameters, such

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as recruitment and mortality. The migration of individuals from adjacent groups can

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influence the magnitude of recruitment estimates; similarly, migration processes can

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bias the determination of natural mortality rates (BEGG e WALDMANN, 1999).

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The main fishing zone of F. paulensis on the continental shelf off the Brazilian

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coast extends from Santos (23°57’S, 46°20’W) to Torres (29°20’S, 49°43’W) (IWAI,

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1973; D’INCAO, 1991). There are likely two main reproductive stocks, which are fishing

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exploitation targets: one at the southern coast (26°54’S, 48°39’W) and another at the

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southeastern coast (23°57’S, 46°20’W) (ZENKER & AGNES, 1977). To date, only two

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studies have analyzed the genetic structure of F. paulensis populations. When studying

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the population structure of F. paulensis with allozymes, GUSMÃO et al. (2005) found

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they were genetically structured in two distinct stocks (south and southeast).

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TEODORO et al. (2015), however, found an absence of genetic structure when

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analyzing the mtDNA Cytochrome c oxidase subunit 1 (COI) region. Such differences

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in results are likely related to the type of molecular marker used in each work, as well

4

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as the sample universe with few sampled localities. In this way, using a molecular

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markers more commonly applied in demographic analyses could help elucidate the

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genetic boundaries of pink shrimp populations.

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Genetic differences among individuals and stocks are the basis for determining

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the degree of reproductive isolation, which is one of the main mechanisms by which

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differences between populations arise (BEGG & WALDMAN, 1999). These genetic

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differences are commonly inferred analyzing the mitochondrial DNA (mtDNA) Control

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Region (D-loop), the most rapidly evolving and highly variable non-coding region in the

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mitochondrial genome (AMARAL et al., 2015). Genomic approaches are being

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increasingly used and provide much larger amounts of data, but since they require

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substantial resources for subsequent bioinformatics, initial knowledge on population

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structure and demographic history of the population obatined using D-loop analysis is

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highly valuable (EXCOFFIER et al., 2009; BOWEN et al., 2014). This marker is a

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useful tool for population analyses at microevolutionary time scales that has been

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effective in the genetic evaluation of shrimp populations (McMILLAN & PALUMBI,

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1995; DUMONT et al., 2009). In fact, this mtDNA marker has been used to successfully

114

elucidate the phylogeography and population structure of several penaeid species (e.g.

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McMILLAN & PALUMBI, 1995; McMILLEN-JACKSON & BERT, 2003; 2004; TZENG et

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al., 2004; TZENG, 2007; DUMONT et al., 2009; CAO & LI, 2016). In this context, the

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aim of this work was to assess and clarify the population genetic structure of the pink

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shrimp F. paulensis in the south-southeast coast of Brazil, through mtDNA control

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region analysis.

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2. MATERIAL AND METHODS 2.1 Sampling

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Sampling of individuals included areas used by the species along its distribution

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at the Brazilian coast (Figure 1.1). Seven sites were sampled along the states of Rio de

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Janeiro (RJ), São Paulo (SP), Santa Catarina (SC) and Rio Grande do Sul (RS),

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covering the main pink shrimp fishing areas. As the presence of F. paulensis at its

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northern distribution limits (Espírito Santo and Bahia coasts) is very rare, sampling was

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not possible in these areas. Fresh material was sampled with a shrimp fishing boat

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equipped with trawl nets with mesh sizes 20 mm and 18 mm. After each trawl, the

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biological material was stored on ice until their transport to the laboratory, where each

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specimen was identified morphologically following COSTA et al. (2003) and TEODORO

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et al. (2016). Each sample was then individually stored in absolute ethanol. The DNA

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yield and quality from individuals stored in 95-100% ethanol are usually equivalent as

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from fresh or frozen tissue (DICK et al., 1993). We also used specimens previously

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cataloged in biological crustacean collections at São Paulo University and Rio Grande

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Federal University.

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Figure 1. Farfantepenaeus paulensis sampling locations along the south-southeast

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coast of Brazil. RJ = Rio de Janeiro state; SP = São Paulo state; SC: Santa Catarina

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state; RS: Rio Grande do Sul state. GUA = Guanabara Bay (RJ); UBA = Ubatuba (SP);

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SAN = Santos (SP); ITA = Itajaí (SC); LAG = Laguna (SC); LPX = Peixe Lagoon (RS);

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LPA = Patos Lagoon (RS).

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2.2 Molecular analysis

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Total genomic DNA was extracted from the abdominal muscle tissue of

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individuals, using a salt extraction protocol adapted from ALJANABI & MARTIN (1997).

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Concentration

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spectrophotometer. Amplification of 659bp of the mtDNA control region was performed

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by Polymerase Chain Reaction (PCR), in a Veriti (Applied Biosystems®) thermocycler,

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using specific primers (Pm690F: 5’ GCTGCTGGCACAAATTTTAGC 3’ and Pm1900R:

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5’ CCTTTTTCAGGCACTTCATT 3’) previously designed at the Marine Molecular

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Ecology Laboratory of the Rio Grande Federal University. PCR reactions were done

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with a total volume of 25 µL, containing ultrapure H2O, PCR Buffer (10%), MgCl2 (3

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mM), 1% BSA solution, dNTPs (0.4 mM), primers (0.2 µM of each), Thermus aquaticus

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polymerase (2.5 U/µL) and 4 µl of DNA (concentration between 150 and 250 ng). PCR

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steps followed DUMONT et al. (2009): 1) initial denaturation of 2’ at 94°C; 2) 10 cycles

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of 10’’ at 94°, 45’’ at 55°C and 1’15’’ at 68°C; 3) 25 cycles of 10’’ at 94°C, 45’’ at 55°C,

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68°C for 1’15’’ – this step was performed adding 10’’ of extension time per cycle,

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starting with 1’15” and ending with 7’15”; and 4) a final extension of 5’ at 68°C.

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Amplified products were submitted to electrophoresis on 1% agarose gels and

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photographed on a UV Transilluminator UVP® M20. These products were purified

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through precipitation with 15% PEG 8000 (Polyethylene Glycol PM 8000) following

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HARTLEY & BOWEN (1996), and a total of 109 purified products were sequenced in

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both directions at Macrogen (South Korea – http://dna.macrogen.com/eng/). After

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molecular analysis, sequences were stored in GenBank (for accession number please

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see supplementary material).

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2.3 Data analysis

(ng)

of

extracted

DNA

was

measured

in

a

Biodrop®2000

7

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Sequences were aligned using Clustal W (THOMPSON et al., 1994) and

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manually edited in BioEdit 7.0.9 (HALL, 1999). The number of haplotypes was

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calculated in DnaSP 4.10.9 (ROZAS & ROZAS, 1999). Haplotype and nucleotide

169

diversities, Analysis of Molecular Variance (AMOVA, using the distance matrix method)

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and the fixation index Phi-st (EXCOFFIER et al., 1992) were calculated using Arlequin

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3.11 (EXCOFFIER et al., 2005). For Phi-st, Jmodel-test version 2.1.10 (DARRIBA et

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al., 2012) was used to identify the substitution model that best fitted our F. paulensis

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data set, which indicated the Tamura & Nei distance method. Preliminary Phi-st

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analyses based on mtDNA control region showed no genetic difference among the

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sampled localities (Phi-st < 0.009). Due to this lack of genetic structure and considering

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geographic proximity, we grouped the dataset by states (RJ, SP, SC, RS), increasing

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the number of individuals per region and consequently the robustness of statistical

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tests.

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A haplotype network was constructed by the Median Joining method (95%

180

Confidence Level) in PopART (BANDELT et al., 1999). To test for isolation by distance,

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a Mantel test correlated Phi-st values and geographic distances (SLATKIN, 1993;

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ROUSSET, 1997). Statistical significance was determined by permutation analysis

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(1000 permutations) with a 95% significance level. Geographical distances were

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estimated by measuring the linear distances between each location along the coastline

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(MCMILLEN-JACKSON & BERT, 2004).

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The demographic history of F. paulensis was evaluated using mismatch

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distribution and raggedness indexes (HARPENDING, 1994). The number of differences

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observed between pairs of haplotypes is usually multimodal in demographically stable

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populations (high raggedness value), and unimodal in populations that have

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experienced recent expansion (low raggedness value) (HARPENDING, 1994). The

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raggedness index was calculated in Arlequin 3.11, and the sum of squared deviations

8

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(SSD) between the observed and the expected mismatch was used to validate the

193

estimated stepwise expansion model (SCHNEIDER E EXCOFFIER, 1999). SSD

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significance (P-value) was obtained through the bootstrap parametric method. In this

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test, if 95% or more of the simulated distribution show a better fit than the observed

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mismatch, the expansion model is rejected (DA SILVA CORTINHAS et al., 2016).

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Tajima's D and Fu's Fs neutrality tests (TAJIMA, 1989; FU, 1997) were also used to

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assess demographic processes: negative values due to excess haplotypes generally

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indicate demographic expansion.

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3. RESULTS

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A total of 99 haplotypes were found for the mtDNA control region of the 109

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individuals of F. paulensis sampled in south-southeast Brazil. Sampled regions showed

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very high haplotype diversity (mean h = 0.9978) and low nucleotide diversity (mean π =

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0.01238), with 92% (N = 91) of unique haplotypes, i.e., found in only one individual.

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The number of haplotypes found for each region, as well as their respective haplotype

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and nucleotide diversities, can be seen in Table 1.

Region

N

H

S

h

π

RJ

13

13

45

1.0000 ± 0.0302

0.068240 ± 0.037714

SP

26

26

51

1.0000 ± 0.0107

0.055491 ± 0.029817

SC

37

34

64

0.9940 ± 0.0086

0.052353 ± 0.027924

9

RS

33

30

70

0.9943 ± 0.0090

208 0.068720 ± 0.036013

Total

109

99

x̅ = 58 ± 12

x̅ = 0.9971 ± 0.0034

209 x̅ = 0.061201 ± 0.008504 210

Table

1.

Genetic diversity of

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the mtDNA control region (D-loop) of Farfantepenaeus paulensis from the four regions sampled

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along the south-southeast coast of Brazil. RJ = Rio de Janeiro; SP = São Paulo; SC = Santa

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Catarina; RS = Rio Grande do Sul; N = number of individuals used in the analysis; H = number

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of haplotypes; S = number of polymorphic sites; h = haplotype diversity; π = nucleotide diversity;

215

x̅ = mean values.

216 217 218 219 220 221

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The haplotype network evidenced a panmitic population pattern (Figure 2), with

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a very high number of haplotypes but few mutations between them, and indicated an

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apparent lack of genetic structure between regions. Only four haplotypes were shared

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by two different regions: H29, observed in SP (Ubatuba) and SC (Laguna); H33,

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observed in one individual from SP (Santos) and three from SC (one from Itajaí and

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two from Laguna); H45, present in SC (Itajaí) and RS (Patos Lagoon); and H71, shared

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by SC (Laguna) and RS (Patos Lagoon).

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Figure 2. Median-Joining network of pink shrimp mtDNA control region haplotypes,

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indicating haplotype distribution along the localities. Each circle represents a haplotype,

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with size being proportional to frequency and colors to sampled region. Black circles

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represent mean vectors, and black lines represent mutational steps. RJ: Rio de

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Janeiro; SP: São Paulo; SC: Santa Catarina; RS: Rio Grande do Sul.

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The Analysis of Molecular Variance (AMOVA) indicated no significant genetic

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difference among individuals from the sampled regions, with intrapopulational variation

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(99.74%) much higher than interpopulational variation (0.26%). Phi-st values based on

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Tamura & Nei distance method also showed no genetic structure, ranging from -

238

0.00920 to 0.01012 (Table 2). The Mantel's test showed no significant relationship

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between genetic distance and geographical distance, indicating that genetic structure

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by distance does not occur (r = -0.07419933, p = 0.6247).

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Table 2. Phi-st values (Tamura & Nei distance method, below diagonal) among

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sampled regions and their respective p-values (above diagonal). RJ: Rio de Janeiro;

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SP: São Paulo; SC: Santa Catarina; RS: Rio Grande do Sul.

RJ

RJ *

SP 0.74414

SC 0.37402

RS 0.71777

SP

-0.00920

*

0.31445

0.30371

SC

0.00203

0.00263

*

0.08789

RS

-0.00833

0.00252

0.01049

*

244 245

Mismatch analysis showed unimodal distributions for all the sampled regions,

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which, associated with high haplotype diversity and low nucleotide diversity, indicates a

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pattern of recent demographic expansion. These results corroborate Tajima's D and

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Fu's Fs neutrality tests (Figure 3, Table 3), which, when negative, indicate demographic

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expansion.

250 251 252

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Table 3. Sudden expansion model parameters and goodness-of-fit tests of the sampled

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pink shrimp populations. SSD = sum of squared deviations; raggedness index;

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corresponding p-values. Locations

RJ

SP

SC

RS

Parameters Tau

8.193

5.668

6.930

7.125

Theta 0

0.262

0.000

0.009

0.000

Theta 1

99999.000

99999.000

140.23400

99999.000

Goodness-of-fit Test SSD

0.00267066 0.01661007 0.00053237 0.01324107

p

0.94100000 0.05600000 0.93700000 0.02800000

Raggedness

0.01084813 0.01420118 0.00817484 0.00798826

p

0.95300000 0.68700000 0.85900000 0.82900000 Neutrality Tests

256 257

Tajima's D

-1.85098

-1.84656

-2.08182

-1.88326

P(D simul < D obs)

0.01700

0.01800

0.00400

0.01100

Fu's Fs

-6.48753

-24.12485

-25.15585

-21.40615

Prob(sim_Fs <=obs_Fs)

0.00200

0.00000

0.00000

0.00000

13

258 259

Figure 3. Pairwise mismatch distributions of pink shrimp populations based on the

260

mtDNA control region, with simulated model of recent expansion; results of Tajima's D

261

test and Fu's Fs and their p-values are also shown for each sampled region.

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4. DISCUSSION

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In this work, we analyzed for the first time the mtDNA control region of F.

264

paulensis along the south-southeastern Brazilian coast (RJ, SP, RS and SC states),

265

detecting very high haplotype diversity and connectivity between regions. Phi-st

266

analysis, AMOVA and the haplotype network showed no significant genetic differences

267

among the sampled regions, and individuals from different regions shared some

268

haplotypes. These combined results indicate that F. paulensis forms a panmitic

269

population along its south-southeastern distribution in Brazil.

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Whereas there are no apparent geographical barriers among the sampled

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regions, the lack of genetic structure found for F. paulensis is likely related to its life

272

cycle and high dispersal capacity of its planktonic larvae. The life history of F. paulensis

273

includes spawning and early larval stages in oceanic waters, with a period (~4-10

14

274

months) of larval/juvenile development in estuarine waters (D’INCAO et al., 2002).

275

These early larval stages exhibit high dispersal capacity, often more related with ocean

276

currents and winds (D’INCAO, 1991) than temperature and salinity (CALAZANS,

277

1978).

278

In the south-southeast coast of Brazil, two major currents could influence the

279

dispersal and consequent gene flow among the studied regions: the Brazil Current,

280

which flows southwards and is more intense in the summer; and the Malvinas (or

281

Falklands) Current, which flows to the north and is more intense during winter

282

(ZAVIALOV et al., 1998). The Malvinas Current flows along the outer Argentinean shelf

283

(STEVENSON et al., 1998), but its coastal branch influences the inner shelf in south

284

Brazil. This coastal branch mixes with fresh waters coming from the La Plata River and

285

the Patos Lagoon, forming the Subtropical Shelf Water (SSW). This water mass flows

286

over the shelf and extends to the Southeast Brazil when south quadrant winds intensify

287

in the winter. During the summer, the SSW converges with the coastal branch of

288

Brazilian Current (CBBC), carrying tropical oligotrophic waters (SILVEIRA et al., 2000).

289

Thus, the southeastern and southern continental shelf of Brazil present alternating

290

dominance of these water masses, favoring larval dispersion from one fishery stock to

291

another both northwards and southwards. These seasonal variations in the

292

directionality of current flow probably contribute to the high connectivity found for F.

293

paulensis populations.

294

The occurrence of fauna from southern cold waters at the coast of Rio de

295

Janeiro has been recorded in Sepetiba Bay (RJ) (STEVENSON et al., 1998), located at

296

the same latitude (22°S) as Guanabara Bay, sampled in this study. Using ecological

297

and satellite data, the authors observed that organisms from colder and less saline

298

oceanic waters from South Brazil can disperse to the north along the inner continental

299

shelf. This could also favor the intense gene flow found for F. paulensis. Recently, high

300

connectivity was detected for populations of the blue crab, which also depends on

15

301

estuaries to complete its life cycle (LACERDA et al., 2017) and for other non-estuarine

302

crustaceans, such as the Barba-Ruça shrimp Artemesia longinaris (CARVALHO-

303

BAPTISTA et al., 2014).

304

Our results corroborated the hypothesis that marine shrimp populations tend to

305

be panmitic (BENZIE, 2000). The magnitude and patterns of genetic structure in

306

marine species are a result of a complex interaction between biogeographical history,

307

gene flow potential and the accomplishment of this potential in the face of possible

308

barriers (AVISE et al., 1987). Population analyses of marine species showing low or no

309

genetic structure are common in species with high potential for larval dispersal

310

(PALUMBI, 2003); unlike terrestrial and freshwater species, the dispersal possibilities

311

of marine animals are generally large due to long larval periods and high reproductive

312

outputs (PALUMBI, 1992). For instance, F. paulensis females can release up to

313

570,000 eggs in their lifetime (IWAI, 1973), which probably contributes to the high gene

314

flow found. The genetic diversity observed for F. paulensis indicates this dispersal

315

promotes gene flow high enough to distribute haplotypes along with the species’

316

occurrence areas, even between geographically distant regions. A pattern of genetic

317

connectivity was found for the mtDNA COI region for F. paulensis (TEODORO et al.,

318

2015) along its distribution, as well as for another Pleoticus muelleri shrimp

319

(CARVALHO-BATISTA et al., 2018). There is no apparent barrier to gene flow among

320

F. paulensis groups in south-southeastern Brazil, and the continuous coast with

321

suitable habitats seems to be a path for short and long-distance larval dispersal.

322

The high haplotype diversity observed for F. paulensis is a common feature for

323

the mtDNA control region of penaeid shrimp (PALUMBI & BENZIE, 1991; MCMILLEN-

324

JACKSON & BERT, 2004). Similar results have been found for the control region of

325

Farfantepenaeus duorarum (MCMILLEN-JACKSON & BERT, 2004), Farfantepenaeus

326

notialis (ROBAINAS-BARCIA & GARCÍA-MACHADO, 2012), and Penaeus monodon

327

(WAQAIRATU et al., 2012). High diversity mtDNA control region diversity was also

16

328

observed for other crustaceans such as the blue lobster Panulirus inflatus (GARCÍA-

329

RODRIGUES & PEREZ-ENRIQUEZ, 2008). On the other hand, TEODORO et al.

330

(2015) found a low diversity of COI haplotypes in F. paulensis, which is likely due to the

331

lower amount of polymorphisms of this marker when compared to the mtDNA control

332

region (DUMONT et al., 2009). The control region, also known as the A-T-rich region,

333

does not code for a functional gene, so it is under fewer functional and structural

334

constraints, resulting in a high average substitution rates (SACCONE et al., 1987).

335

Hypervariability is one of the most evident features of the mtDNA control region in pink

336

shrimp species, and high diversity seems to be characteristic of decapod crustaceans

337

(MCMILLEN-JACKSON & BERT, 2004; NARO-MACIEL et al., 2011; SINGH et al.,

338

2019). Similar results of genetic connectivity with a history of population expansion

339

have been also reported in other marine crustaceans (BENZIE et al., 2002; NARO-

340

MACIEL et al., 2011; SINGH et al., 2019), and the mtDNA control region has been

341

used to elucidate intra specific population structure of several crustaceans (e.g. GUO

342

et al., 2012; LAURENZANO et al., 2013; AMARAL et al., 2015; AZUMA & CHIBA,

343

2017; SINGH et al., 2019). This pattern of high haplotype diversity in the mtDNA

344

control region is also common in migratory fish species exhibiting large panmictic

345

populations (TURNER et al., 2002; ELY et al., 2005; SANTOS et al., 2007).

346

The absence of genetic structure observed for F. paulensis was also found by

347

TEODORO et al. (2015) when analyzing the mtDNA COI region. However, when

348

studying the population structure of F. paulensis with allozymes, GUSMÃO et al. (2005)

349

found they were genetically structured in two distinct stocks, south and southeast. This

350

discrepancy in results is probably related to the used marker together and the few

351

localities sampled in the study of GUSMÃO et al (2005). Events such as sudden

352

climatic changes and other natural or anthropogenic disturbing factors are independent

353

of the biological properties of the species (ALLISON et al., 2003), but can affect the

354

temporal stability of the enzymatic systems in natural populations, and consequently

17

355

their genetic characteristics (BARCIA et al., 2005). For instance, high fishing pressures

356

can favor the evolution of characteristics related to the life history of the exploited

357

species, either due to the high mortality caused or due to the selectivity of fishing gear

358

size (LAW, 2000). Therefore, analysis of allozymes may not have detected actual

359

genetic variability, since several factors can affect the temporality of allelic frequencies

360

(BARCIA et al. 2005).

361

Demographic analyses indicated unimodal distributions for all the sampled

362

regions, supporting a model of recent demographic expansion. A sudden population

363

growth also probably influenced the high genetic diversity found for F. paulensis.

364

During these growth events, the rate of stochastic loss of haplotypes decreases,

365

overriding genetic drift (MCMILLEN-JACKSON & BERT, 2004). Low nucleotide

366

diversity combined with high haplotype diversity is generally attributed to a

367

demographic expansion that occurred after a reduction in effective population size,

368

retaining new mutations (AVISE, 1994; DUMONT et al., 2009). Considering both

369

Tajima's D and the haplotype and nucleotide diversities, we can infer that the unimodal

370

mismatch distribution of our sampled populations is due to recent demographic

371

expansions. This is probably related to a recovery process initiated after the fishing

372

collapse faced by the populations in the late 1990s (D'INCAO et al., 2002). During a

373

bottleneck event in a large and relatively stable population with high haplotype

374

diversity, we often assume there is loss of genetic diversity (CARSON, 1990). Although

375

haplotype diversity generally decreases, the remaining haplotypes can be highly

376

divergent due to the genetic diversity present in the previous population (ALLCOCK &

377

STRUGNELL, 2012). The high mutation rate combined with the rapid life cycle, high

378

population recovery capacity and the lack of barriers to gene flow likely contributed to

379

the development and maintenance of the high haplotype genetic diversity of F.

380

paulensis, even after the fishery collapse.

18

381

Along with the lifecycle and genetic traits of F. paulensis, changes in closed

382

season policies after the fishery collapse have likely contributed to the recovery

383

process and genetic diversity of the species. In 1996, the Patos Lagoon Forum created

384

new rules on artisanal fishing for this main estuary used by F. paulensis, with the

385

publication of an Ordinance by the Brazilian Institute of the Environment and

386

Renewable Natural Resources (IBAMA) (D'INCAO & REIS, 2002; KALIKOSKI &

387

VASCONCELLOS, 2003). The main changes were the establishment of a closed

388

period specific to the region, the regulation of fishing gear and the requirement of an

389

environmental license provided by IBAMA to exploit resources from the lagoon

390

(D'INCAO & REIS, 2002; KALIKOSKI & VASCONCELOS, 2003). Also, the

391

geographical range of the closed season limits increased after 1996 to include from the

392

coast of Espírito Santo to Rio Grande do Sul (FRANCO et al., 2009). When the closed

393

season was created for pink shrimp, other penaeid species (such as the seabob shrimp

394

Xiphopenaeus kroyeri) continued to be exploited and eventually captured juveniles of

395

Farfantepenaeus. From 2002 the closed season became valid for all exploited penaeid

396

species (FRANCO et al., 2009), protecting these accidentally captured juveniles and

397

allowing them to complete their life cycles. Considering the high genetic connectivity

398

found among localities, we can infer that changes in the legislation from both south and

399

southeast Brazil have contributed to the beginning of the recovery process of F.

400

paulensis populations. Additionally, the collapse of the stocks during the ‘90s shifted

401

the fishing effort from shrimp to demersal fish and the fleet migrated to deeper waters,

402

searching for alternative resources (D’INCAO et al., 2002). The change of focus on the

403

fishery effort, associated with the changes in closed season policies, likely allowed the

404

expansion process of pink shrimp populations.

405

The high connectivity observed with mtDNA suggests that estuaries provide

406

"stepping stone" areas for migration, allowing gene flow. Thus, more importantly than

407

considering populations as a single genetic stock and applying the same management

19

408

measures, we reinforce the need to integrate management according to the intrinsic

409

attributes of each estuary used by F. paulensis. Management must be specific to each

410

region considering their different dynamics and fishery pressures, and the fact that high

411

mortality rates of pink shrimp stocks in these environments may lead to decreased

412

gene flow between them. The continued genetic monitoring of the F. paulensis

413

populations is fundamental to understand how exploited populations respond after a

414

fishery collapse. Genetic studies (mtDNA, nuclear and genomic analysis), associated

415

with morphometric studies and fishery statistics, can provide the basis needed for the

416

proper management of F. paulensis. Additionally, the study of other exploited penaeids,

417

especially F. brasiliensis and F. subtilis, is important for understanding the

418

consequences of overfishing on pink shrimp species.

419

5. CONCLUSION

420

The analysis of the mtDNA control region indicates that F. paulensis forms a

421

panmitic population along its south-southeastern distribution in Brazil, with a pattern of

422

recent demographic expansion. This scenario is a result of a complex interaction of

423

several factors, from the influence of ocean currents on larval dispersal to the effect of

424

a possible genetic bottleneck caused by overfishing. The consequences of

425

management of a specific area are likely to be reflected in all other regions used by F.

426

paulensis. Management measures such as the offseason period can contribute to the

427

recovery process of the populations. The knowledge on the diversity and genetic

428

structure of F. paulensis obtained in the present study is a fundamental part of the

429

scientific basis necessary to inform decision makers for the elaboration of adequate

430

management strategies of pink shrimp populations.

431

6. ACKNOWLEDGMENTS

432

This work was conducted during a scholarship provided to SSAT from CAPES –

433

Brazilian Federal Agency for Support and Evaluation of Graduate Education. Additional

20

434

support came from CNPq – Brazilian National Research Council, which provided

435

research fellowships to SSAT (SWP #312448/2015-5), RCC (PQ #304784/2011-7),

436

MCP (PQ #312470/2018-5) and LFCD (INCT-Mar COI, #56062/2010-7). We

437

acknowledge the Department of Biology (Faculty of Philosophy, Sciences and

438

Languages – CCDB/FFCLRP/USP) and the Museum of Zoology of the São Paulo

439

University, as well as the Laboratory of Decapod Crustaceans, for providing samples

440

from their Biological Crustacean Collections. The authors are thankful to Dr. Marcos

441

Alaniz Rodrigues and Prof. Dra. Helena Lavrado for providing valuable samples for this

442

work; to lab members from LABCAM, LCD and LEMM for their help during fieldwork,

443

laboratorial analysis and data interpretation, which helped to improve this manuscript.

444

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Highlights for manuscript

- The mtDNA control region of F. paulensis is analyzed for the first time - F. paulensis showed high connectivity along its main distribution areas in Brazil - Populations experienced recent demographic expansion - Overfishing and lifecycle contribute to these patterns of connectivity and demography - This information should be considered in management strategies of pink shrimp stocks

Rio Grande, October 13, 2019 To the Estuarine, Coastal and Shelf Science editorial office Dear Editor, We are submitting the original research manuscript “High genetic connectivity among pink shrimp Farfantepenaeus paulensis (Pérez-Farfante, 1967) groups along the south-southeastern coast of Brazil” to be considered for publication in Estuarine, Coastal and Shelf Science after revisions. We declare that this manuscript has not been published and is not under consideration for publication elsewhere, and we declare no conflicts of interest. Sincerely, Dr. Sarah de Souza Alves Teodoro, and on behalf of co-authors Corresponding author, email [email protected]