Molecular Phylogeny of Western Atlantic Farfantepenaeus and

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Molecular Phylogenetics and EvolutionVol. 18, No. 1, January, pp. 66–73, 2001doi:10.1006/mpev.2000.0866, available online at http://www.idealibrary.com on

Litopenaeus Shrimp Based on Mitochondrial 16S Partial SequencesRodrigo Maggioni,* Alex D. Rogers,* Norman Maclean,† and Fernando D’Incao‡

*School of Ocean and Earth Science, Southampton Oceanography Centre, University of Southampton, European Way, SouthamptonSO14 3ZH, United Kingdom; †Division of Biodiversity and Ecology, School of Biological Sciences, University of Southampton,

Southampton SO16 7PX, United Kingdom; and ‡Laboratorio de Crustaceos Decapodos, Departamentode Oceanografia, Universidade do Rio Grande, C.P. 474, 96201-900 Rio Grande, Brazil

Received October 27, 1999; revised July 24, 2000; published online December 12, 2000

have recently reviewed the systematics of the suborder

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Partial sequences for the 16S rRNA mitochondrialgene were obtained from 10 penaeid shrimp species:Farfantepenaeus paulensis, F. brasiliensis, F. subtilis,F. duorarum, F. aztecus, Litopenaeus schmitti, L. se-tiferus, and Xiphopenaeus kroyeri from the westernAtlantic and L. vannamei and L. stylirostris from theeastern Pacific. Sequences were also obtained from anundescribed morphotype of pink shrimp (morphotypeII) usually identified as F. subtilis. The phylogeny re-sulting from the 16S partial sequences showed thatthese species form two well-supported monophyleticclades consistent with the two genera proposed in arecent systematic review of the suborder Dendro-branchiata. This contrasted with conclusions drawnfrom recent molecular phylogenetic work on penaeidshrimps based on partial sequences of the mitochon-drial COI region that failed to support recent revisionsof the Dendrobranchiata based on morphologicalanalysis. Consistent differences observed in the se-quences for morphotype II, coupled with previousallozyme data, support the conclusion that this is apreviously undescribed species of Farfantepenaeus.© 2000 Academic Press

INTRODUCTION

The shrimps of the Family Penaeidae are knownaround the world as valuable resources for fisheriesand aquaculture in tropical and subtropical regions(Neal and Maris, 1985; Provenzano, 1985). Despitetheir commercial importance, many aspects of the bi-ology of the Penaeidae are poorly understood and thereis still much debate about fundamental issues, such assystematics and population dynamics (Dall et al.,990). The systematics of the group, in particular, hasot yet been fully resolved, even for the heavily ex-loited and widely studied species of the genusenaeus (sensu Perez-Farfante, 1969, used throughout

the present paper). Perez-Farfante and Kensley (1997)

1055-7903/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

66

Dendrobranchiata and have proposed that the fivesubgenera of the genus Penaeus be raised to genericstatus.

Biochemical approaches to the study of the system-atics and population genetics of the Penaeidae, usingallozymes, has previously led to the conclusion that themorphological similarity of the group is mirrored by alow level of genetic polymorphism (e.g., Lester, 1979;Redfield et al., 1980; Mulley and Latter, 1980; Sundennd Davis, 1991; Benzie et al., 1992; Tam and Chu,993). However, Palumbi and Benzie (1991) discoveredigh levels of molecular divergence between Penaeustylirostris, P. vannamei, P. esculentus, and Metap-naeus endaevori, as revealed by partial sequencing of2S and COI mtDNA regions. The discovery of suchariability in DNA sequences and the recent availabil-ty of more efficient DNA-based techniques have gen-rated a surge of new molecular work on Penaeus (e.g.,arcia et al., 1996; Ball et al., 1998; Tassanakajon etl., 1998; Moore et al., 1999; Xu et al., 1999).Among the most recent work, Baldwin et al. (1998)

have approached the phylogeny and biogeography ofPenaeus shrimps through the partial sequencing of themitochondrial COI gene. They concluded that molecu-lar data from this gene does not support the systematicrevisions of Perez-Farfante and Kensley (1997) basedon thelycum condition. The thelycum is the femaleexternal reproductive structure and its morphology iscrucially involved in reproductive strategy. When aspecies has a closed thelycum, spermatophores can beimplanted only after molting when the exoskeleton isstill soft. When the thelycum is open this is not the caseand mating usually occurs toward the end of the moltcycle before ecdysis (Dall et al., 1990). The function ofthis external reproductive structure and its ecologicalrelevance give the thelycum great importance as ataxonomic character. Perez-Farfante (1969) proposedthe subdivision of the genus Penaeus into four subgen-era, including Litopenaeus, which was diagnosed by an

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67MOLECULAR PHYLOGENY OF WESTERN ATLANTIC SHRIMP

open thelycum. The fact that the molecular data gath-ered by Baldwin et al. (1998) failed to support theurrent proposed taxonomy (Perez-Farfante and Ken-ley, 1997) was surprising and raises an interestinghylogenetic question. It is apparent that additionalequence data is required to resolve contrasting con-lusions of morphological studies and those based onhe mitochondrial COI gene.

In the present paper, the phylogeny of western At-antic prawns, from the genera Farfantepenaeus anditopenaeus, is reconstructed through partial sequenc-

ng of the 16S mitochondrial region. The status of aorphotype of pink shrimp from the northeastern Bra-

ilian coast usually identified as F. subtilis is alsonvestigated. Morphological variation within F. subti-is, previously recorded by Perez-Farfante (1969),long with more recent biochemical genetic evidencendicates that this sympatric morphotype is in facteproductively isolated from F. subtilis (D’Incao et al.,

1998).

MATERIAL AND METHODS

Sampling

Ten species were screened in the present study, 5Farfantepenaeus, 4 Litopenaeus, and the species Xi-phopenaeus kroyeri, which was used as an outgroup forthe phylogenetic analysis (see Tables 1 and 2). In ad-dition to these species, we studied a previously unde-scribed morphotype of pink shrimp, usually identifiedas F. subtilis, which will be simply referred to as “mor-photype II.”

All samples consisted of a piece of tail muscle pre-served either in 99% ethanol or in 6 M urea, 1% sar-cosyl, 10 mM NaPO4, pH 6.8. F. paulensis, F. brasil-iensis, F. subtilis, L. schmitti, and X. kroyeri wereacquired from fishermen or from fresh fish markets ateight sampling sites along the Brazilian coast between33°S and 2°S (Table 1; see also Fig. 1), during June1999. For the most southern sampling site F. paulensis

Sample Sizes and Locations for the BraziliaF. subtilis, F. brasiliensis, Litopenae

F. paul. morph. II

Rio Grande (32°S) 2Florianopolis (28°S) 2Guaratuba (25°S)Santos (24°)Vitoria (20°S) 2Recife (8°S) 2Fortaleza (4°S) 4Sao Luıs (2°S)Total 4 8

was supplied by the Carcinology Laboratory of theUniversity of Rio Grande (Rio Grande, Brazil). F.

uorarum, F. aztecus, and L. setiferus were caught inharleston Harbor (33°N) by researchers of the Southarolina Department of Natural Resources in May999. In addition to western Atlantic species, L. van-amei and L. stylirostris from the eastern Pacific werelso sampled. L. vannamei was obtained from the ex-erimental culture ponds of the Marine Shrimp Labo-atory of the Federal University of Santa CatarinaFlorianopolis, Brazil), and L. stylirostris was obtainedrom fisherman off the coast of Ensenada, Baja Cali-ornia (32°N). Finally, four samples of morphotype IIere the same specimens used in a previous allozyme

tudy (D’Incao et al., 1998), and they were first sam-led at Fortaleza (4°S, Ceara, Brazil) along with F.ubtilis (see Fig. 1). In this case whole individuals haveeen preserved for 7 years in 70% ethanol.

CR and Sequencing

DNA was extracted through digestion of a smalliece of muscle in 100 mM Tris–HCL, pH 8.0, 1.25%DS, and 390 ng/ml proteinase K (approx. 0.012

units/ml of Boehringer Mannheim, Cat. No. 1373-196).The 400-ml preparations were incubated for 3–4 h at55°C in a water bath and then a standard phenol/chloroform–isoamyl alcohol extraction was carried outwith precipitation of DNA by 2:1 ice-cold 100% ethanolplus 1:10 3 M sodium acetate (600 mM final concentra-tion). The 20-ml PCRs were performed in 0.5-ml tubes,using ultrapure PCR water, and contained 200 mMeach dNTP, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2.5mM MgCl2, 1 mM each primer, 1 unit Taq, and 10 to 50ng template DNA. The primers used to amplify the 39end of the 16S rRNA mitochondrial gene were the16Sar (CGC CTG TTT ATC AAA AAC AT) and 16Sbr(CCG GTC TGA ACT CAG ATC ACG T); their positionsin the human genome are 2510 and 3080, respectively(Simon et al., 1994; Palumbi 1996). PCR was performedeither on a Perkin–Elmer 480 or on a Hybaid PCR-

hrimp Species Farfantepenaeus paulensis,schmitti, and Xiphopenaeus kroyeri

F. subt. F. bras. L. schmi X. kroy.

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Phylogenetic Analysis

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68 MAGGIONI ET AL.

Express thermocycler and comprised a 94°C/4 min ini-tial denaturing step followed by 30 cycles of 94°C/1min, 52°C/1 min, and 72°C/1 min. A final elongationstep of 72°C/10 min was used. Oil overlay or hot lidworked equally well. PCR products were purified withQiagen Qiaquick PCR Purification columns (Cat. No.28106) according to manufacturers guidelines. The10-ml cycle-sequencing reactions were prepared usingPerkin–Elmer BigDye Terminator Ready Reactionmixes (Cat. No. 4303152), using the concentrations,amounts, and thermocycling conditions recommendedby the manufacturer. The products of cycle-sequencingwere purified through Qiagen DyeEx Spin kits (Cat.No. 63104) and screened in a Perkin–Elmer ABI 377automated sequencer. All samples were double-checked by reverse sequencing.

FIG. 1. Sampling sites for the Brazilian coast prawns used in theresent study. Codes as follows: su, Farfantepenaeus subtilis; pa, F.

paulensis; mII, morphotype II; br, F. brasiliensis; ch, Litopenaeusschmitti; xi, Xiphopenaeus kroyeri. Also shown is the distribution ofF. subtilis and F. paulensis, according to Perez-Farante (1969) (F.

aulensis distribution includes modifications by D’Incao, 1995), ba-hymetry, and circulation pattern (according to Johns et al., 1990;

SEC, South Equatorial Current; NBC, North Brazil Current; BC,Brazil Current).

Sequences were aligned using Clustal X (Thompsonet al., 1997) and double-checked by eye. A consensussequence for each species and morphotype II wasdrawn from the initial set. The consensus sequencesretain the variable sites, which were very small innumber in this study. Reported sequences for F. notia-lis (detailed in Machado et al., 1993) and P. monodon(K. H. Chu, J. G. Tong, and T. Y. Chan, unpublished;GenBank Accession No. AF105039) were also includedin the analysis. All phylogenetic analyses, as well asbasic statistics, were performed using PAUP* 4.0 beta4a (Swofford, 1998). Three methods of tree buildingwere used: maximum-likelihood, maximum-parsi-mony, and neighbor-joining. For all methods, tree to-pology was evaluated by bootstrapping of the originaldata set. For maximum-likelihood and maximum-par-simony, a heuristic search was employed and startingtrees were always obtained by random sequence addi-tion. Tree visualization and drawing were carried outusing TreeView version 1.5 (Page, 1996).

Maximum-likelihood analysis was performed underthe General Time-Reversible (GTR) model of base sub-stitution. The Modeltest 3.0 (Posada and Crandall,1998) algorithm was used to evaluate the choice of GTRmodel, which produced the most significant log-likeli-hood values, among various models tested. Site-specificsubstitution rates (SSR) and discrete approximation ofthe gamma (G) distribution were used in two separateanalyses to correct for the among-site variation in thesubstitution rates. On GTR 1 SSR analysis, substitu-tion rates were calculated considering the structuralanalysis by Machado et al. (1993), which divided the 39end of the 16S gene into two domains: domain A, withhigher substitution rates, corresponded to sites 209–371 in our sequences. Base frequencies, substitutionrates for the six different substitution types, and rela-tive rates for the two domains considered, or the shapeparameter for the G distribution (a), were estimatedfrom the data set through maximum-likelihood, con-sidering the topology of an initial neighbor-joining tree.The estimated values were used in searches with 1000sequence addition replicates and in the subsequentbootstrappings, which consisted of 100 replicates with100 sequence additions per replicate.

On maximum-parsimony analysis, gaps were consid-ered as meaningful characters and multistate sites onthe consensus sequences were considered polymorphic.The tree presented was found through a search with1000 sequence additions. Bootstrapping of the maxi-mum-parsimony tree consisted of 1000 bootstrap rep-licates, each with 100 sequence addition replicates.Finally, a neighbor-joining tree was constructed fromthe distances calculated under the GTR 1 SSR modelwith the same parameters used for the maximum-

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69MOLECULAR PHYLOGENY OF WESTERN ATLANTIC SHRIMP

likelihood analysis. The topology of the neighbor-join-ing tree was evaluated by 1000 bootstraps.

RESULTS

Variability of Sequences

The partial 16S rRNA mitochondrial gene sequencesobtained comprised fragments of 485 to 488 bp of the 39end of the gene. All the original sequences were depos-ited in GenBank under the Accession Nos. AF192051 toAF192093 and AF255054 to AF255057. Average com-position of the fragments studied were A 5 32.6%, C 513.2%, G 5 21.6%, and T 5 32.6% (when F. notialis and

. monodon sequences are included: A 5 32.9%, C 53.0%, G 5 21.3%, and T 5 32.8%). Significant differ-nces in base composition across all taxa were notetected (x2 5 6.80; df 5 36; P . 0.999). Neverthe-

less, there was a bias in segment composition towardA 1 T that has been observed in other arthropodan

itochondrial gene sequences (Simon et al., 1994). Theomain A of Machado et al. (1993) concentrates onverage almost two-thirds of the absolute number ofubstitutions in a one-third stretch of the sequence. Onhe other hand, loops and stems presented on average2 and 58% of the pairwise substitutions from a total of34 and 259 sites, respectively. On stems, compensa-ory substitutions are observed in a number of sites, asbserved by Machado et al. (1993). The secondarytructure of loops and stems have been tentativelyefined by following Palmero et al. (1988) and Machadot al. (1993).

Table 2 presents the average pairwise GTR 1 SSRistances matrix for the studied 16S sequences. Mor-hotype II shows a degree of genetic divergence from F.ubtilis comparable to the pairwise distances between. subtilis, F. aztecus, and F. paulensis and much

Penaeidae: Pairwi

F. sub. m II F. paul. F. azt. F. duo. F.

F. subtilis (8) 0.002Morphotype II (8) 0.040 0.004F. paulensis (4) 0.043 0.051 0.000F. aztecus (3) 0.044 0.056 0.043 0.000F. duorarum (3) 0.054 0.062 0.057 0.053 0.000F. notialisb 0.062 0.068 0.067 0.062 0.004F. brasiliensis (2) 0.065 0.072 0.059 0.050 0.055 0L. vannamei (3) 0.099 0.081 0.104 0.091 0.095 0L. stylirostris (4) 0.085 0.080 0.099 0.087 0.085 0L. schmitti (8) 0.121 0.113 0.132 0.113 0.113 0L. setiferus (3) 0.114 0.107 0.125 0.106 0.106 0P. monodonc 0.131 0.130 0.141 0.131 0.122 0X. kroyeri (4) 0.183 0.182 0.183 0.178 0.166 0

a Average pairwise GTR 1 SSR distances (sample sizes in parentb Distances calculated between consensus sequences and 16S sequc Distances calculated between consensus sequences and 16S Gen

igher than those between F. duorarum and F. notialisnd between L. schmitti and L. setiferus (Table 2). Inddition, an insertion of three T’s was observed nearhe 59 end of the sequence (sites 29–31), in a regiontherwise conserved among the species studied. Ac-ording to the model for Artemia, this region corre-ponds to a loop in the secondary structure of the 16SRNA (Palmero et al., 1988).

hylogenetic Analyses

Two basic topologies resulted from the tree searchethods described (Fig. 2). They showed considerable

greement, the only major difference among them be-ng the internal branching of the genus Litopenaeus.igure 2A presents the strict consensus topology re-ulting from the maximum-parsimony analysis, onhich two equally parsimonious trees were produced.ootstrap values for this tree were high among differ-nt genera but often low within genera.The majority rule consensus tree resulting from

eighbor-joining was identical to the maximum-parsi-ony tree. Maximum-likelihood produced an alterna-

ive branching for Litopenaeus (Fig. 2B). Branch sup-ort followed a pattern similar to that of maximum-arsimony, with good support for Farfantepenaeus anditopenaeus but poor resolution among species withinhe genera. However, GTR 1 G maximum-likelihoodootstrap shows poor support for Litopenaeus. We ob-erved that an increase in bootstrap replicates and inhe sequence additions per replicate increased theranch support for this model, but significant improve-ent in this testing would require a large amount of

omputer power, not available to us at the presentime.

Summarizing the information on Fig. 2, there istrong support for Farfantepenaeus and for Litope-

Distances Matrixa

t. F. bras. L. van. L. sty. L. sch. L. set. P. mon. K. kro.

2 0.0009 0.098 0.0000 0.084 0.038 0.0011 0.110 0.071 0.067 0.0018 0.103 0.066 0.065 0.009 0.0006 0.111 0.125 0.113 0.134 0.135 —3 0.173 0.163 0.165 0.190 0.178 0.161 0.000

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morphotype II, were evaluated. An allozyme study was

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70 MAGGIONI ET AL.

naeus. Within the genus Farfantepenaeus, a lack ofresolution resulted in a polytomy among F. brasilien-sis, F. dourarum–F. notialis, and the remaining spe-cies. However, the group formed by F. aztecus, F. pau-lensis, F. subtilis, and morphotype II was supported.Within the genus Litopenaeus, the data did not provideenough information to resolve the relationships be-tween the Pacific and the Atlantic species, but L. schi-mitti and L. setiferus clearly form a monophyleticgroup.

DISCUSSION

Morphotype II

In the western Atlantic, six species of Farfante-penaeus and two species of Litopenaeus have been de-scribed. There are an additional two and three species,respectively, in the eastern Pacific. Both Farfante-penaeus and Litopenaeus are endemic to the Americas,except for F. notialis, which occurs on the Atlanticcoast of Africa, and no species occur on both sides of theAmericas (Dall et al., 1990; Perez-Farfante and Kens-ley, 1997). During the present study the genetic rela-tionships among all western Atlantic species, including

FIG. 2. Topologies resulting from the phylogenetic methodssed. (A) Strict consensus tree resulting from the two most-parsimo-ious trees obtained through heuristic search with 1000 sequenceddition replicates (L 5 240; CI 5 0.733; RI 5 0.668; RC 5 0.490).ootstrap support shown for maximum-parsimony (MP) and neigh-or-joining (NJ). Branch lengths correspond to maximum-parsimonynalysis. (B) Maximum-likelihood tree obtained after an heuristicearch with 1000 sequence addition replicates under the GTR 1 SSRodel. Bootstrap branch support shown for two models of sequence

ubstitution. Distribution codes: SCA, Caribbean and/or Southmerica; NCA, Gulf of Mexico and North America; WA, North toouth America; EP, Eastern Pacific; IP, Indo–Western Pacific.

previously carried out on F. paulensis, F. subtilis, andmorphotype II from Brazil by D’Incao et al. (1998).Forty samples of pink shrimp consisting of 21 genuineF. subtilis and 19 of a second, reproductively isolatedgroup designated as morphotype II were analyzed fromFortaleza (4°S, Ceara, Brazil). Morphotype II pos-sessed adrostral and dorsolateral sulci that resembledF. paulensis (F. D’Incao, unpublished observations).Across the 18 allozyme loci studied, morphotype II wasfound to be genetically more similar to F. paulensis(Nei’s 1978 genetic identity, I 5 0.985) than to F.subtilis (I 5 0.947). In addition, morphotype II sharedno alleles with F. subtilis at a polymorphic phospho-gluconate dehydrogenase (enzyme number 1.1.1.44,IUB, 1984) locus (D’Incao et al., 1998). The presence ofa diagnostic locus in sympatric populations is a strongindication of reproductive isolation.

The 16S mitochondrial sequence data in the presentstudy also support the specific status of morphotype II.This putative species showed little sequence diversityacross a wide geographical range among samples fromVitoria (20°S, Espırito Santo, Brazil), Recife (8°S, Per-nambuco, Brazil), and Fortaleza (4°S; refer to Fig. 1),as well as fixed sequence differences from the remain-ing species of Farfantepenaeus in the present study.Morphotype II presents a level of genetic divergencefrom the most closely related species (0.04–0.06) whichis comparable or higher than that between other well-characterized species in this group (Table 2). The 16Smitochondrial gene is considered to be relatively con-served and estimates of divergence of 4–6% have beenrecorded among species of the same genera for otherarthropoda (Simon et al., 1994). However, in contrastto the previous findings with allozyme studies (D’Incaoet al., 1998), the 16S sequences of morphotype II aremore similar to F. subtilis than to F. paulensis. Thesympatric distribution and level of genetic divergencebetween morphotype II and both F. subtilis and F.paulensis indicate that morphotype II is reproductivelyisolated from these species. Furthermore, the geneticdivergence of morphotype II from the remaining west-ern Atlantic species suggests that this animal is anundescribed species of Farfantepenaeus.

Alternatively, this organism could be (1) an occa-sional hybrid or (2) an introduced exotic species. If it isan occasional hybrid, it would have to be assumed thatan extensive and continuous hybridization process wastaking place between species (hybrid swarms—seeGardner, 1997). This is not supported by molecularevidence, as maternal inheritance of mitochondrialDNA would produce sequence agreement with otherFarfantepenaeus species. As for the second hypothesis,there are no records of any attempt to culture exoticFarfantepenaeus in northeastern Brazil, but the intro-duction of exotic larvae in tanker ballast water cannotbe ruled out (e.g., Carlton, 1985). Finally, Perez-

Farfante (1967, 1969) had already recorded the pres-

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71MOLECULAR PHYLOGENY OF WESTERN ATLANTIC SHRIMP

ence of a different morphotype of P. subtilis across itsange of distribution, but attributed this to environ-ental influences producing phenotypic variation.

hylogenetic Reconstruction

According to the review by Dall et al. (1990), theamily Penaeidae is assumed to have had an Indo–est Pacific origin during the late Triassic, with the

arliest fossil record of Penaeus from the Jurassic,aking this group the oldest taxon among the Penaei-

ae. Schram (1982) also supports a Triassic origin ofamily Penaeidae. Together, these studies suggest thathe extant Penaeidae form a monophyletic group, withPenaeus shrimp as a common ancestor. Other groupsay also be considered as ancestral to the Penaeidae

ut there is no fossil evidence to support this (Burken-oad, 1983). However, it must be noted that the fossilecord for crustaceans is poor, especially for those withoorly calcified, soft exoskeletons (Schram, 1982).Based on allozyme and zoogeographic data, Dall et

l. (1990) suggest that most penaeid genera must haverisen in the last 20 My and that a large number ofenaeus species may have originated less than 2 MyP. Sequence data from the COI gene support theypothesis of Dall et al. (1990) that the genus Penaeusrose in the Indo Pacific, based on taxa from this areahowing the deepest mitochondrial DNA lineages andhe highest mitochondrial DNA diversity (Baldwin etl., 1998). COI data also support the radiation ofenaeus westward into the eastern Atlantic and east-ard into the eastern Pacific and western Atlantic. Thenal elevation of the Central America isthmus, datedo between 3.1 and 3.5 My BP (Kennett, 1982) created

barrier to gene flow between eastern Pacific andestern Atlantic populations and probably led to spe-

iation.Phylogenetic reconstruction by Baldwin et al. (1998),

ailed to support the revision of the genus Penaeus byerez-Farfante and Kensley (1997). This was largelyecause the genera Farfantepenaeus and Litopenaeus,eparated on the basis of thelycum condition, were notupported as monophyletic groups by the phylogenyased on COI sequences. However, the monophyletictatus of these genera is well supported in the presenttudy by the 16S mitochondrial DNA sequence datarom the western Atlantic and the two eastern Pacificpecies. The phylogenetic reconstructions based on 16Sequences (Fig. 2) clearly agree on this point and sup-ort the revision of the genus Penaeus by Perez-arfante and Kensley (1997). They also agree in otherays with this morphological classification. For exam-le, western Pacific P. monodon fall outside the groupormed by Farfantepenaeus and Litopenaeus (Fig. 2).uch agreement seems to extend to the level of specificelationships: Perez-Farfante (1969) points to closeorphological similarity between L. setiferus and L.

936), between F. duorarum and F. notialis, andmong F. aztecus, F. subtilis, and F. paulensis (refer toig. 2). Mulley and Latter (1980) and Tam and Chu

1993) have previously presented allozyme data thatre in agreement with other subdivisions of Penaeusroposed by Perez-Farfante and Kensley (1997).Relationships within the genus Farfantepenaeus are

either clear nor well supported. Our findings agreeith those of Baldwin et al. (1998) that F. paulensisppears to have arisen after divergence of F. brasilien-is and F. duorarum, but show a lack of resolutionegarding F. brasiliensis and F. duorarum–F. notialis.he monophyly of F. duorarum and F. notialis istrongly supported. The similarity among the se-uences of these two species is so high that specifictatus could not be supported solely on the basis of the6S gene. Finally, it appears that F. aztecus, F. pau-ensis, F. subtilis, and morphotype II have some sup-ort as a monophyletic group. However, the poor boot-trap support may suggest a recent radiation involvinghese species.

volution of Brazilian Farfantepenaeus

The amplitude and frequency of climatic change in-reased from the late Pliocene, reaching a maximumhroughout the Quaternary, when many glacial eventsaused large sea level oscillations (e.g., Kennett, 1982).herefore, the assumption made by Dall et al. (1990),

hat lowered sea level must have had a significantffect in increasing separation among populations ofhe shallow-water penaeids, seems plausible. Analyz-ng Caribbean fossil coral reefs, Fairbanks (1989) dem-nstrated that around 18,000 years BP, during the lastlacial maximum, the sea level in the western Atlanticas approximately 120 m below current levels. On therazilian coast the narrow and steeply sloping north-astern platform is likely to have been largely aboveea level during much of the Quaternary. This mayave intensified the isolation of the coastal populationsetween the north and the south coasts of Brazil (refero Fig. 1). In addition, in this coastal region the Southquatorial Current splits into two arms, one flowingorthward, forming the North Brazil Current, and an-ther flowing southward, forming the Brazilian Cur-ent (Pickard and Emery, 1982; see Fig. 1). This pat-ern was probably already established in the MioceneKennett, 1982; Hodell and Kennett, 1985). Sea levelariations coupled with the ocean circulation patternay be sufficient to explain the isolation and specia-

ion of the southern F. paulensis. F. paulensis is apecies adapted to colder water and to lower levels ofalinity (Perez-Farfante, 1969) and therefore it mayave originated as an isolated southern populationuring the late Pliocene. Radiation in western Atlantic

Farfantepenaeus, as suggested by 16S phylogeny, could

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72 MAGGIONI ET AL.

have been a consequence of periodic habitat fragmen-tation in the last 2–3 million years.

CONCLUSIONS

Phylogenetic conclusions based on one or even a fewmitochondrial genes may be misleading (Cummings etal., 1995). In contrast with COI data presented byBaldwin et al. (1998), phylogenetic reconstructionbased on 16S partial sequencing in the present papersupports the monophyletic status of the generaFarfantepenaeus and Litopenaeus, as described by

erez-Farfante and Kensley (1997), at least amongestern Atlantic species. The revision of the taxonomyf the genus Penaeus by Perez-Farfante and Kensley1997) is also supported in other ways by 16S data.owever, agreement between 16S and COI data is

lear in some cases, such as where both genes suggestmore recent origin of F. paulensis than F. duorarum

nd F. brasiliensis. Finally, a previously undescribedpecies of the genus Farfantepenaeus has been identi-ed.

ACKNOWLEDGMENTS

The authors thank Amy Ball, from the Marine Resources ResearchInstitute, South Carolina Department of Natural Resources, for thecollection of northern Atlantic samples and cross-checking of se-quence data at a crucial point of the project; Rodolfo Petersen, fromthe Marine Shrimp Laboratory (Laboratorio de Camaroes Marinhos- U.F.S.C., Florianopolis, Brazil), for the samples of L. vannamei andhe help during the field work; Dr. L. F. Navarro for the Mexicanamples of L. stylirostris; and Professor P. Shooligan-Jordan androfessor P. Holligan for the use of facilities at the School of Biolog-

cal Sciences and the School of Ocean and Earth Sciences. Dr. A. D.ogers acknowledges the support of N.E.R.C. Advanced Researchellowship GT5/97/4/MAS during the period of the present study. R.aggioni acknowledges the help and support from friends and col-

eagues during the field trip in June 1999. R. Maggioni is assistantecturer in the Department of Natural Sciences of the Universidadestadual do Ceara and is currently supported by a Ph.D. grant from.N.Pq. (Brazil).

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