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Genetic and Morphological Variation of Northeast Pacific Panopea Clams:Evolutionary ImplicationsAuthor(s): Axayácatl Rocha-Olivares, Luis E. Calderon-Aguilera, Eugenio Alberto Aragón-Noriega,Nancy C. Saavedra-Sotelo and Victor Manuel Moreno-RiveraSource: Journal of Shellfish Research, 29(2):327-335. 2010.Published By: National Shellfisheries AssociationDOI: http://dx.doi.org/10.2983/035.029.0207URL: http://www.bioone.org/doi/full/10.2983/035.029.0207

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GENETIC AND MORPHOLOGICAL VARIATION OF NORTHEAST PACIFIC

PANOPEA CLAMS: EVOLUTIONARY IMPLICATIONS

AXAYACATL ROCHA-OLIVARES,1* LUIS E. CALDERON-AGUILERA,

2

EUGENIO ALBERTO ARAGON-NORIEGA,3NANCY C. SAAVEDRA-SOTELO

1

AND VICTOR MANUEL MORENO-RIVERA2

1Molecular Ecology Laboratory, Department of Biological Oceanography, CICESE, Carretera Ensenada-Tijuana No. 3918, Ensenada, Baja California 22860, Mexico; 2Fisheries and Coastal Ecology Laboratory,Department of Marine Ecology, CICESE Carretera Ensenada-Tijuana No. 3918, Ensenada, BajaCalifornia 22860,Mexico; 3Centro de Investigaciones Biologicas del Noroeste, Unidad Guaymas, Km 2.35Camino al Tular, Estero de Bacochibampo, Guaymas, Sonora 85454, Mexico

ABSTRACT Geoduck clams have become the most profitable emerging fishery resource in Northwest Mexico, with profits of

more thanUS$30million during the last few years. The fishery targets two species—Panopea globosa in the Gulf of California and

Panopea generosa on the Pacific coast of Baja California—but is managed indistinctively. Despite its growing importance,

scientific research on the basic biology of the Mexican stocks has been inexistent until recently. A major gap in knowledge is the

interspecific distinction in structural and functional biological attributes. Consequently, the aim of this article is to provide the

biological basis of phenotypic (morphometric) and genetic distinction between P. globosa and P. generosa to assist in their

management and conservation.We found thatP. generosa from the Pacific coast of Baja California is significantly smaller thanP.

globosa from the northernGulf of California in shell length, width, and height (t-tests,P < 0.0001), and that shell width and height

scale differently to length in both species. Multivariate analyses (multidimensional scaling) provided additional support (stress¼0.04) to the species and geographical distinction. Genetic data from the nuclear ribosomal DNA provided contrasting results

between polymerase chain reaction– restriction fragment length polymorphisms and direct sequencing. Ribosomal DNA

sequences revealed higher diversity (haplotype and nucleotide) in P. globosa. Standing in sharp contrast with the low intraspecific

divergence, was the very large genetic differentiation between species in excess of 20% corrected Kimura 2-parameter sequence

divergence and accounting for 98%of the molecular variance of both species. This differentiation was found to be of consequence

for novel methods of molecular species identification and for the interpretation of the phylogeography and evolution of Panopea

in the North Pacific. The relevance of our findings goes to the heart of filling a major information gap pertaining to the distinction

of both species. Scientific and lay stakeholders of these valuable resources need to ascertain and acknowledge this distinction to

adopt sustainable management and exploitation practices.

KEY WORDS: geoduck clam, morphometrics, genetic divergence, ribosomal DNA, Baja California, Panopea

INTRODUCTION

Geoduck clams in the genus PanopeaMenard, 1807, are largeinfaunal bivalves burrowing from 0.6–1 m into sediments, at wa-ter depths ranging from the intertidal to 110 m. These clams are

renowned for their extreme longevity, reaching a record 168 y inage (Bureau et al. 2002).Among filter-feeding burrowing bivalves,the geoduck is one of the largest; its shell reaches more than 25 cm

and the siphon is more than 1 m in length (Goodwin & Pease1987). Panopea has a worldwide antitropical distribution, andthree species are found in the North Pacific: Panopea japonica

Adams, 1850, off the coasts of China, Japan and Korea; Panopeagenerosa Gould 1850 from Alaska to Baja California, Mexico;and Panopea globosa Dall, 1898, which is endemic to the Gulfof California, Mexico (Fig. 1). Since 1984, P. generosa has been

erroneously referred to as P. abrupta, a fossil species incorrectlymade synonymous with P. generosa (Vadopalas et al. 2010). Inthe Northeast Pacific, a fishery for the Pacific geoduck clam (P.

generosa) began in the 1970s off the coast of Washington and, 5 ylater, in British Columbia. Since then and until 2004, nearly all ofthe world’s supply of this highly valued product originated from

that area (Orensanz et al. 2004). Since the early 2000s,Mexico hasincreasingly contributed to the Northeast Pacific geoduck fisheryand, by 2006, the yearly catch was around 1,200 t, which is on par

with that from neighboring United States and Canada (Fig. 2).However, unlike the monospecific catches of these countries, the

geoduck fishery in Mexico is sustained by P. generosa from theeast coast of Baja California and P. globosa from the Gulf ofCalifornia, but has been managed indistinctively. The fishery has

attracted considerable and increased interest given its high de-mand and profitability. For instance, from 2006 to 2008 alone,combined harvests from both coasts of the Baja California

peninsulaweremore than 1,200 t/y and represented a total annualincome of approximately US$30 million (SAGARPA 2007).

In sharp contrast with the extensive amount of publishedand unpublished research on P. generosa from Canada and the

United States (see Feldman et al. (2004) for a review), thescientific information on the Mexican populations of Panopeaspp. has only begun to be produced. Basic biological studies such

as patterns of distribution and abundance, age, growth, re-production, population structure, and morphological and ge-netic variation are urgently needed to assess the dynamics and

connectivity of these populations subject to exploitation. Theresearch needs to encompass both species and the resultsanalyzed comparatively to identify relevant biological differencesamong the species leading to their recognition as distinct fishing

units. Notably, only three publications are available on the basicbiology of P. globosa in Mexico: two from Bahıa de Guaymas-Empalme, Sonora (Central Gulf of California), and one from the

Upper Gulf of California (Calderon-Aguilera et al. 2010). The*Corresponding author. E-mail: arocha@cicese.mx

Journal of Shellfish Research, Vol. 29, No. 2, 327–335, 2010.

327

first reports on the morphometric variation and reproductivebiology of samples collected during 2004 and 2005, featuringa predominance of large clams and documenting peak spawningbetween January and February (winter), at the time of lowest sea

surface temperature (18�C) (Aragon-Noriega et al. 2007). Thesecond is a complementary report focusing on the reproductiveaspects of the same sample collections, but elaborating on a com-parative analysis with the reproductive cycles ofP. generosa from

Figure 2. Landings of geoduck clams. Panopea generosa in Canada (British Columbia) and the United States (Washington), and P. generosa and

P. globosa inMexico. Sources:Ministry of Environment, Oceans &Marine Fisheries, Canada (www.env.gov.bc.ca/omfd); www.st.nmfs.noaa.gov (US);

and www.conapesca.sagarpa.gob.mx (Mexico).

Figure 1. Geographical distribution of Panopea spp. clams in the North Pacific. P. japonica, thick continuous line off Asia; P. generosa, thick broken

line off North America: P. globosa, thin patterned line in the Gulf of California and southwestern Baja California. The presence of P. globosa off the

Pacific coast of Baja California needs scientific corroboration. (Inset) Sampling localities ofP. generosa (Islas Coronado and Bahia de SanQuintin) and

P. globosa (San Felipe and Puerto Penasco).

ROCHA-OLIVARES ET AL.328

Canada and P. zelandica from New Zealand (Arambula-Pujolet al. 2008). The third and most recent paper deals with the ap-

parent synchrony between larval development and the peak ofprimary productivity in the Upper Gulf of California, as pre-dicted byCushing’smatch–mismatch hypothesis (Cushing 1975).Through the analysis of time series, the authors show that

gametogenic development of P. globosa is triggered by a steepdecrease in temperature (Calderon-Aguilera et al. 2010).

The goal of this article is to provide fundamental informa-

tion on the levels of morphological and genetic variation amongspecies of Panopea off the Mexican coasts. Structural and func-tional distinctionof both species is required to provide a biological

basis for their proper management and conservation. In addition,our results also offer a means of molecular species identificationthat can be applied to food products as well as a phylogeo-graphical framework for addressing the evolution and biogeog-

raphy of these commercially important clams.

MATERIALS AND METHODS

Sample Collection

We analyzed clams collected in the subtidal from prospective

fisheries of Panopea carried out on the Pacific coast of BajaCalifornia (P. generosa: Isla Coronado and San Quintin) and inthe Gulf of California (P. globosa: San Felipe and Puerto

Penasco; Fig. 1, Table 1). They were sampled along belt tran-sects perpendicular to the coast, following a random designstratified by suitable soft-bottom clam habitat. Depth rangedfrom 10–25 m. Clams were extracted by divers using a water jet

to dig them up, and the clams were analyzed immediately uponlanding. The shell was removed from the tissue, and valves weremeasured to the nearest millimeter using calipers. We measured

shell length (SL; straight-line distance between the anterior andposterior margins of the shell), shell height (SH; distance be-tween the dorsal and ventral shell margins), and shell width

(SW; distance between closed left and right valves with ventralmargins touching). A siphon tissue sample was dissected andpreserved in 95% ethanol for genetic analyses.

Genetic Analyses

After rinsing the tissue sample in sterile deionized water,total genomic DNA was extracted from of each organism using

proteinase digestion and incubation in Chelex 100 (Bio-Rad,Hercules, CA) (Walsh et al. 1991). Briefly, a small piece of tissue(0.1 g) was incubated at 55�C for 1 h in 500 mL Chelex 100 (Bio-

Rad, Hercules, CA) (10%) and proteinase K (0.6 mg/mL), andsubsequently at 95�C for 15 min to kill proteinase activity. The

nuclear ribosomal DNA (rDNA) region encompassing ITS1,5.8SRNA, and ITS2 genes was polymerase chain reaction (PCR)amplified using ITS4 (5#-TCC TCC GCT TAT TGA TAT GC-3#) and ITS5 (5#-GGAAGTAAAAGTCGTAACAAGG-3#)primers (White et al. 1990). Reactions (25 mL) contained 0.18mMdNTPs, 13 PCRbuffer (10mMTrisHCl, 50mMKCl, and1.5 mM MgCl2), 0.4 mM each primer, 1 U Taq DNAPol (New

England Biolabs, Ipswich, MA), and 2 mL DNA extraction(approximately 50–100 ng). Thermal cycling consisted of 1min at94�C, followed by 36 cycles of 1min at 94�C, 1min at 55�C, and 2min at 72�C,with a final extension of 10min at 72�C.Quality andquantity of PCR products were assessed by agarose (1.5%) gelelectrophoresis stained with ethidium bromide (0.5 mg/mL).DNA sequence variation in nuclear rDNA was assessed by

restriction fragment length polymorphisms (RFLPs), in whichPCR products were digested with 5 restriction enzymes (Cfo I,Rsa I, Hae III, Taqa I, and Dpn II) chosen to have recognition

sequences based on available sequence data (P. generosa: Gen-bank accession no. EF035123). Digestions were carried outfollowing the manufacturer’s protocols (New England Biolabs,

Ipswich, MA), and PCR-RFLP patterns were resolved byelectrophoresis in 2% agarose gels stained with ethidium bro-mide (0.5 mg/mL). The PCR-RFLP haplotypes resolved by these

five restriction enzymes were subsequently sequenced. Briefly,PCRproducts were purified by digestion with ExoSAP-IT (USB,Cleveland, OH) before sequencing using PCR primers supple-mented by nested primers ITS2 and ITS3 (White et al. 1990) with

BigDye v3.1 chemistry and run in a ABI377 DNA sequencer(Applied Biosystems, Foster City, CA) following the manufac-turer’s protocols.

Data Analyses

Mean SL, SW, and SH, and SW-to-SL and SH-to-SL ratios

were compared among species using t-tests with STATISTICA7.1 (Statsoft). Nonmetric multidimensional scaling (MDS) wasused to assess the morphometric structure of clams in multidi-mensional space. MDS was implemented on a Bray-Curtis

similarity matrix of log-transformed variables using PRIMER6 (Clarke & Gorley 2006).

RFLP restriction patterns for each enzyme were used to infer

the number of restriction sites, which were binary coded forpresence or absence to construct a composite haplotype includingall enzymes. PCR-RFLP data were analyzed with the program

REAP 4.0 (McElroy et al. 1992). Sequence data were firstassembled, checked for sequencing artifacts, and end trimmedfrom poor-quality base calls with the program Codon Code

Aligner 3.0 (CodonCode Corporation). Individual sequenceswere aligned using Clustal X 2 (Thompson et al. 1997) usingdefault parameters. Sequences were used to compute haplotypeand nucleotide diversity indices, and we assessed the partitioning

of molecular variance between and within species with ananalysis of molecular variance (AMOVA) as implemented inArlequin 3.01 (Excoffier et al. 2005). Phylogenetic relationships

among haplotypes were estimated using the neighbor-joiningalgorithm, and branch support was assessed by nonparametricbootstrap with MEGA 4 (Tamura et al. 2007). The phylogenetic

relationships among P. generosa, P. globosa and P. japonicawereassessed using a publicly available ortholog sequence from thelatter (Genbank accession no. AB377638).

TABLE 1.

Comparison of mean morphometric characters of Panopeaspp. from Baja California.

Character P. generosa P. globosa t Value P Value

SL 137.4 (18.4) 155.9 (18.6) –5.23 <<0.0001

SW 83.0 (11.0) 105.2 (12.7) –9.70 <<0.0001

SH 53.1 (13.0) 74.96 (10.9) –11.11 <<0.0001

SW/SL 0.61 (0.05) 0.68 (0.06) –6.52 <<0.0001

SH/SL 0.39 (0.07) 0.48 (0.05) –7.61 <<0.0001

Data are presented as mean (SD) in millimeters. SL, shell length (largest

axis); SH, shell height (largest dorsal–ventral measurement); SW, shell

width (body thickness of both valves).

VARIATION IN PANOPEA CLAMS FROM NORTHWEST MEXICO 329

RESULTS

Morphometric Differentiation

Based on the three morphometric variables (SL, SW, SH),our data reveal that P. globosa was consistently and signifi-cantly larger than P. generosa (Table 1). In addition, differences

in the two dimensionless ratios (SW/SL, SH/SL) indicate thatshell width and height scale differently to shell length in bothspecies, pointing to distinct interspecific allometric growth

(Table 1). This distinction is also readily observed in the MDSplot, which reveals a clear demarcation between P. generosa (nand:) andP. globosa (3 and *) at a very low stress value (Fig.

3). Interestingly, although large P. globosa organisms from theGulf of California mostly overlap in the bidimensional plot,smaller P. generosa clams from the two localities in the Pacific

coast of Baja California segregate in the morphometric spaceprojected on these 2 axes (Fig. 3).

Genetic Differentiation

Molecular amplification of the rDNA of geoduck clams wassuccessfully carried out in all but three specimens, producing anapproximate 1,000 base pair (bp) fragment (Table 2). Digestion

with five restriction endonucleases revealed very low levels ofintraspecific variation in the ribosomal genes of both species. Allenzymes but one (Rsa I) were monomorphic in P. generosa,

producing only two composite haplotypes present in both local-ities, whereas only 1 haplotype was scored in P. globosa (Table 2).Standing in sharp contrast to the intraspecific diversity was the

very large differentiation between species. Binary coding ofinferred restriction sites revealed that, except for the polymorphicRsa I site of P. generosa, no other site was shared between species,which made these data unsuitable to estimate levels of divergence.

A set of six specimens from each locality, encompassing the3 PCR-RFLP haplotypes, were directly sequenced. After endtrimming poor-quality data, rDNA sequences resulted in four

haplotypes for P. generosa (945–949 bp), of which only one wasshared between localities (PGENIC02), and eight for P. globosa(984–986 bp), of which two were shared between sites

(PGLOPP01, PGLOPP02). Contrary to the diversity patternsuggested by PCR-RFLPs, rDNA sequences revealed that P.globosa had a higher molecular diversity than P. generosareflected in more haplotypes (8 vs. 4 haplotypes), higher

haplotype (0.94 vs. 0.68) and nucleotide (0.0059 vs. 0.0008)diversities, and a higher mean divergence (0.006 vs. 0.001; Table

2). The last two reflected the very incipient differentiation amongintraspecific haplotypes featuring only three segregating sites andtwo indels in P. generosa, and 22 segregating sites and one indelin P. globosa (bold type in Fig. 4). Consequently, although P.

generosa haplotypes were close phylogenetically, two divergentP. globosa sequences (PGLOSF03 and PGLOSF05) were respon-sible for the higher levels of intraspecific divergence and nucleo-

tide diversity (Table 2, Fig. 4)Interspecific divergence revealed by rDNA sequences was as

dramatic as the one shown by PCR-RFLPs. The sequences

diverged considerably, reaching a corrected (Kimura 2 param-eter (Kimura 1980)) divergence of 0.21 (uncorrected P ¼ 0.18),standing in sharp contrast to the mean intraspecific divergencein both species. Not included in these estimates of divergence

were two large (approximately 50-bp) indels fixed in eachspecies (indicated in italics in Fig. 4). Consequently, AMOVArevealed that the vast majority of the molecular variance was

allocated interspecifically (FCT ¼ 0.98, P ¼ 0.0001), whereasa smaller but nonsignificant fraction occurred between sam-pling localities within species (FSC ¼ 0.089, P ¼ 0.15). The

phylogenetic reconstruction including all three North PacificPanopea species (P. generosa, P. globosa, and P. japonica) basedmostly on ITS-1 and 5.8S rDNA, revealed the close evolution-

ary relationship between P. japonica (endemic to the NorthwestPacific) and P. generosa (ranging from Alaska to the Pacificcoast of Baja California). P. globosa (endemic to the Gulf ofCalifornia) represented a sister divergent lineage (Fig. 5). A close

inspection of the rDNA sequences revealed that the sister–taxonrelationship ofP. generosa andP. japonica is supported by a largenumber of synapomorphies (see asterisks in Fig. 4) relative to P.

globosa. As expected, most of the interspecific variation occurredin the internal transcribed spacers (ITS-1 and ITS-2; Fig. 4).

DISCUSSION

This article is the first to document major phenotypic andgenetic differentiation of P. generosa and P. globosa currently

under exploitation in Mexico.

Morphological Variation

The two species of geoduck analyzed in this study showedcontrasting patterns of geographical morphometric variation.The mean size of P. globosa was geographically heterogeneous,

differing between northern and central populations of the Gulfof California, whereas P. generosa SL was remarkable homo-geneous in samples fromMexico, Washington state, and British

Columbia (Fig. 6).Panopea globosa from the northern Gulf of California (San

Felipe and Puerto Penasco) were significantly larger (SL, 155.9mm vs. 147.7 mm; t ¼ 2.8, P ¼ 0.005) than those measured by

Aragon-Noriega et al. (2007) in a comparable sample (n ¼ 77)collected in 2004/2005 from the Empalme Bay, on the easternshore of themidGulf of California (Fig. 6). This variation could

result from sampling biases. Samples from the northern gulfwere collected deeper (10–25m) than those from the central gulf(8–12 m); however, our sampling strategy does not allow us to

assess the existence of bathymetric segregation of size classes ineither of the 2 localities. Alternatively, this contrast could reflectdifferences in the size structure of allopatric populations, which

Figure 3. Nonmetric MDS plot of morphometric variables of samples of

Panopea generosa (Islas Coronado (IC) and San Quintin (SQ)) and P.

globosa (San Felipe (SF) and Puerto Penasco (PP)).

ROCHA-OLIVARES ET AL.330

may be influenced by significantly heterogeneous environmen-tal conditions along the Gulf of California (Lluch-Cota et al.

2007).On the other hand, the mean size of P. generosa clams col-

lected from the Pacific coast of Baja California (SL, 137.4 mm)did not differ significantly from that of conspecifics fromCanada

(SL, 139.0 mm; British Columbia (Bureau et al. 2002)) and theUnited States (SL, 135.2 mm; Puget Sound, WA (Goodwin &Pease 1991); Fig. 6). Even though sample sizes in this study (P.

generosa: n¼ 65;P. globosa: n¼ 71)may be judged insufficient toassess the morphological variation of both species, they are notfar from the reasonable sample size of n � 80 suggested for the

assessment ofmean phenotypic traits inP. generosa (Campbell &Rajwani 1998). In concert with this, we found that the measure-ments of twoP. generosa samples fromBaja California (n¼ 65 intotal) harbor as much size variance as that of historical data sets

encompassing three orders of magnitude more data fromWashington state (n ¼ 11,154) and British Columbia (n ¼14,184) (Goodwin & Pease 1991, Bureau et al. 2002). Neverthe-

less, we do not question the need for additional geographical andtemporal samples to assess the phenotypic variability ofMexicanPanopea spp. populations along the Pacific coast of Baja

California and in the Gulf of California.Remarkable size differences were found between both species.

The extent to which they relate to biological species-specific

differences (e.g., in size at age or in the age structure of sampledpopulations) or to sampling biases (e.g., introduced by fisherstargeting different depths and regions of spatially/depth-structuredpopulations) is unclear. However, homogeneity of variances in

samples fromP. generosa andP. globosa (compare SDs in Table 1)points to an adequate representation of the underlying distribu-tion of morphometric characters, and our field observations do

not suggest the existence of strong microgeographical structureswithin aggregations. The scope of phenotypic variation in thepopulations of both species remains undersampled in Mexico;

however, the structural (i.e., size) differences revealed in ourstudy are indicative of differences in individual- and population-level rate processes such as growth, reproduction, and recruit-ment, which are at the core of population dynamics. In the case

of P. globosa in the Gulf of California, the reproductive cyclemay play a significant role in maintaining these phenotypic dif-ferences. Because Panopea clams are fundamentally temperate

bivalves (Feldman et al. 2004), reproduction of P. globosa in theGulf of California is tightly coupled and triggered by a decrease

in water temperature during winter, a few months before thepeak in primary production (Aragon-Noriega et al. 2007;Calderon-Aguilera et al. 2010). Compared with other geoduckpopulations of P. generosa and P. zelandica at higher latitudes,

this environmental constraint entails a more protracted non-reproductive period (up to 7–8 mo) (Aragon-Noriega et al. 2007;Calderon-Aguilera et al. 2010), during which energy can be

directed to somatic growth. In contrast, samples of P. generosafrom the Pacific coast of BajaCalifornia show a pattern of partialprotracted spawning during the year (Calderon-Aguilera, un-

published results) comparable with those of conspecifics at higherlatitudes (Andersen 1971, Sloan & Robinson 1984). Such dif-ferences in life history attributes, which are likely related to theirdeep evolutionary divergence (see discussion in the next section),

in concert with differences in physical and biological environ-mental variables between the coastal Pacific and the Gulf ofCalifornia habitats (Sandoval-Castillo et al. 2004) are likely re-

sponsible for the phenotypic interspecific differentiation.

Genetic Patterns of Differentiation and Phylogeography

Previous genetic studies of geoduck clams were aimed atdesigning hypervariable microsatellites and using them along

with less polymorphic allozymes to assess levels of populationgenetic heterogeneity in beds from Canada (British Columbia)and Washington state (Vadopalas & Bentzen 2000, Vadopalaset al. 2004, Miller et al. 2006). This is the first study addressing

intra- and interspecific levels of genetic variation using rDNAsequences in 2 species of Panopea.

The clearly and unequivocally distinct PCR-RFLPs patterns

between P. generosa and P. globosa represent a powerful ap-proach for species identification, in need of corroboration andvalidation from a broader survey. In fact, our morphological,

RFLP, and DNA sequence data provide evidence of concordantand significant differentiation betweenP. generosa andP. globosa.The RFLP differentiation was so extreme that the species didnot share any of the inferred restriction sites, making the data

unsuitable for quantitative analyses (McElroy et al. 1992), butcompletely adequate and informative for the determination ofspecies identity. DNA-based methods of molecular identification

TABLE 2.

Molecular diversity of rDNA genes in species of Panopea spp. from Mexico.

Species Collection Geographical Position Date n

PCR-RFLP DNA Sequence

Haplotype Frequency d A h p ns

Panopea generosa Isla Coronado 32�25#N, 117�16#W April 2008 32 AAAAA 15 0.001 2 0.53 0.0000* 6

ABAAA 15

San Quintin 30�33#N, 115�56#W May 2008 33 AAAAA 10 3 0.60 0.0015 6

ABAAA 23

Panopea globosa San Felipe 31�01#N, 114�50#W April 2008 39 BCBBB 39 0.006 5 0.93 0.0087 6

May 2008

Puerto Penasco 31�19#N, 113�36#W February 2008 32 31 5 0.93 0.0025 6

Sample size (n), mean intraspecific corrected divergence (d ¼ K2P (Kimura 1980)), number of haplotypes (A), haplotypic diversity (h), nucleotide

diversity (p), number of organisms sequenced (ns). Haplotypes refer to restriction patterns of Cfo I, Rsa I, Hae III, Taqa I, and Dpn II.

* The seemingly contradictory result of a null nucleotide diversity of this sample in the presence of 2 haplotypes results from the fact that both

haplotypes (PGENIC01 and PGENIC02) only differ by 1 insertion/deletion (indel), which is not incorporated into the computation of p.

VARIATION IN PANOPEA CLAMS FROM NORTHWEST MEXICO 331

Figure 4. Alignment of consensus rDNA sequences (partial 18S, ITS-1, 5.8S, partial ITS-2) of Panopea spp. from Baja California (P. generosa and

P. globosa) and Japan (P. japonica, Genbank accession no. AB377638). Nucleotides marked with an asterisk represent synapomorphies between

P. generosa and P. japonica, relative toP. globosa. Two large indels in ITS-1 ofP. japonica andP. globosa are in italics. Polymorphic nucleotides within

species are indicated in bold type; positions with intraspecific indels are indicated by a lowercase underlined bold letter.

ROCHA-OLIVARES ET AL.332

have bloomed in many fields, including food and forensic science

(Teletchea et al. 2005). Our data suggest that PCR-RFLP ofrDNAwith any of the 4 intraspecificallymonomorphic restrictionenzymes (Cfo I,Hae III,Taqa I,Dpn II) could provide an efficient

and cost-effective method of species identification of thesePanopea species. Even thoughwe have only tested it inP. generosaand P. globosa, we expect the method to be extendable to more

species, as has been shown for other bivalves such as clams(Fernandez et al. 2001) and scallops (Lopez-Pinon et al. 2002).

Levels of intraspecific differentiation assessed by PCR-RFLPswere inadequate and biased compared with those obtained from

DNA sequences. RFLPs are expected to reveal a fraction of nu-cleotide substitutions found in DNA sequences, particularly inslow-evolving genes (Birt et al. 1995). However, we did not expect

to find such contrasting patterns in the levels of intraspecificpolymorphisms between PCR-RFLP and DNA sequencing,because they generally provide congruent results (Bernatchez &

Danzmann 1993, Slippers et al. 2002). In light of the very low

levels of detected variation, the usefulness of PCR-RFLPs of

rDNA for studying the genetic structure of Panopea may beseverely limited.

Sequences of the nuclear rDNA provided a more accurate

picture of the levels of inter- and intraspecific genetic variation.Direct sequencing revealed the presence of only 1 PCR productand no intraindividual polymorphisms, as has been shown in

the rDNA of other bivalves (Insua et al. 2003). Low levels ofrDNA sequence variation have limited the study of intraspecificgenetic structure in a number of bivalves, such as in the marineclam Mya arenaria (ITS1 (Caporale et al. 1997)) and the ocean

quahog Arctica islandica (ITS1 and ITS2 (Dahlgren et al.2000)). On the other hand, despite the limited polymorphismsand shallow divergences, ITS-1 variation was able to detect

significant genetic discontinuities in the phylogeography of thefreshwater bivalveLasmigona subviridis (ITS1 (King et al. 1999)).The species of Panopea displayed contrasting levels of genetic

diversity (very high in P. globosa and low in P. generosa), but

Figure 5. Unrooted neighbor-joining phylogram based on Kimura 2-parameter distances of rDNA (ITS-1 and 5.8S) sequence data of North Pacific

Panopea spp. clams.

Figure 6. Comparison of mean shell length (+95%CI) for populations ofPanopea generosa from the Northwest coast of Baja California,Mexico (Pgen

MX, this study); Puget Sound, WA (Pgen WA (Goodwin & Pease 1991)); and British Columbia, Canada (Pgen BC (Bureau et al. 2002)); and

populations of Panopea globosa from the northern (Pglo NG, this study) and central (Pglo CG (Aragon-Noriega et al. 2007)) Gulf of California.

VARIATION IN PANOPEA CLAMS FROM NORTHWEST MEXICO 333

consistent low levels of sequence divergence among rDNAsequences. Given the limited number of samples and localities

analyzed, we cannot infer geographical patterns of geneticvariation, and the levels of diversity should not be generalizedto the entire species. For instance, testing the possible presence ofan edge effect as the cause of depressed genetic variability in the

peripheralP. generosa populations of BajaCaliforniawill requireassessing genetic diversity of northern populations with rDNAsequences. Moreover, the usefulness of rDNA sequences to

address intraspecific levels of genetic structure remains unclear.On the other hand, the sequences provided valuable data forcomparative interspecific analyses, including data for an addi-

tional species (P. japonica).Sequence evolution of rDNA was significant among the

3 species of Panopea, revealing genetic differentiation in excessof 20% corrected sequence divergence and accounting for 98% of

molecular variance inP. generosa andP. globosa. Significant levelsof divergence in the internal spacers of rDNA have been foundin other bivalves such as oysters (Reece et al. 2008) and scallops

(Insua et al. 2003). To our knowledge, the systematics of extantPanopea species has not been quantitatively assessed. AvailablerDNA sequence data allowed us to advance a phylogenetic

hypothesis of the relationships among P. generosa, P. globosa,andP. japonica. The sister–taxon relationship betweenP. generosaand P. japonica relative to P. globosa among North Pacific

Panopea is an unsuspected finding that is extremely well supportedby numerous synapomorphies in the rDNA sequence data (Fig.4). This hypothesis unveils intriguing biogeographical implicationsfor the evolution of Panopea. If this finding is corroborated by

additional data, such as DNA sequences from additional nuclearand mitochondrial genes as well as morphological characters, itwould imply that the geographical proximity of Northeast Pacific

P. generosa andP. globosa is unrelated to a recent shared ancestry.Instead, our data suggest that speciation of Panopea in the North

Pacific may have involved trans-Pacific dispersal or vicariancefollowed by subsequent reproductive isolation betweenP. japonicaand P. generosa lineages. This evolutionary trajectory has beenhypothesized for other antitropical organisms such as fishes of the

genus Sebastes (Rocha-Olivares et al. 1999a, Rocha-Olivares et al.1999b). Without additional taxa, the evolutionary affinities ofP. globosa can only be speculated. A thorough discussion of the

phylogeography of Panopea is beyond the scope of this article andwill be presented elsewhere; nevertheless, our results provide thefirst evidence of a significant biological and evolutionary distinc-

tion ofP. generosa andP. globosa that needs to be incorporated intheir management and conservation.

To our knowledge, the distinction and taxonomic validity ofP. generosa and P. globosa have not been scientifically chal-

lenged. Nevertheless, the relevance of our findings transcendsthe biological and evolutionary issues discussed earlier, andgoes to the heart of filling major knowledge and information

gaps pertaining to the distinction of both species. Scientific andlay stakeholders of these valuable resources need to ascertainand acknowledge this distinction to adopt sustainable manage-

ment and exploitation practices.

ACKNOWLEDGMENTS

This study was funded by the Baja California State Secretaryof Fisheries and Aquaculture. A Consejo Nacional de Ciencia yTecnologıa grant (no. 651-208) supported the sabbatical leave

of E. A. A. N. at Centro de Investigacion Cientıfica y deEducacion Superior de Ensenada and of L. E. C. A. at Centrode Investigaciones Biologicas del Noroeste.

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