Molecular markers used to analyze species-specific status in

abalones with ambiguous morphology.

ABSTRACT Pacific abalone abalone (ăbəlō`nē), popular name in the United
States for a univalve gastropod mollusk of the genus Haliotis, members of which
are also called ear shells, or sea ears, as their shape resembles the human ear.
..... Click the link for more information. (Haliotis discus hannai) and Californian
red abalone (Haliotis rufescens) are easily distinguished by their shell and
epipodial characteristics. A few individuals of H. rufescens were discovered to
have epipodial coloration similar to that of H. discus hannai suggesting that they
may have been hybrids of the two species, because hybridization
hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.

2. molecular hybridization

3.
..... Click the link for more information. of these species has been informally
reported from local hatcheries. The present study uses molecular genetic methods
to determine whether the rarely colored variants represented hybrids between the
two species. Two hundred RAPD RAPD Randomly Amplified Polymorphic DNA
RAPD relative afferent pupillary defect (ophthalmology; aka Marcus-Gunn
Pupil) primers were tested with PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction

Polymerase chain reaction (PCR)

..... Click the link for more information. on two DNA DNA: see nucleic acid.
DNA
or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic
compound found in all living cells and many viruses. It is the chemical substance
of genes. pools of eight individuals of each species. Primers that showed different
amplification profiles between species were tested on 27 randomly sampled
individuals of each species to check the existence of polymorphisms. Two primers
differentiate both species. Primer 356 amplified an approximately 450 bp and 460
bp specifics-DNA fragments in H. discus hannai that were not present in red
abalone, whereas primer 368 amplified a 750 bp, 850 bp, 860 bp, and 1,190 bp
specific-DNA fragments in H. rufescens that were not present in H. discus hannai.
The amplification pattern of the DNA of individuals with ambiguous morphology
for both primers was characteristic of red abalone. The results strongly suggested
that the specimens with unusual epipodial coloration were not hybrids, but rather,
phenotypic variants of H. rufescens. Future studies should focus on the cause of
the variation of epipodium color in H. rufescens, which could be either genetic
polymorphism polymorphism, of minerals, property of crystallizing in two or
more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing
as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are
brookite, anatase (or octahedrite), and rutile. or phenotypic plasticity.

KEY WORDS: abalone, hybridization, Haliotis rufescens, Haliotis discus hannai,

DNA testing DNA testing
Analysis of DNA (the genetic component of cells) in order to determine changes
in genes that may indicate a specific disorder.

Mentioned in: Acoustic Neuroma, Retinoblastoma, Von Willebrand Disease ,

RAPD, culture, Chile

INTRODUCTION

Abalones are gastropod gastropod, member of the class Gastropoda, the largest
and most successful class of mollusks (phylum Mollusca), containing over 35,000
living species and 15,000 fossil forms. molluscs belonging to the genus Haliotis
(Mollusca: Gastropoda). These molluscs have a dorsally flattened bowl-shaped
shell that protects most of the body, and a large ventral foot only slightly smaller
than the shell. A major feature of the exterior morphology is the epipodium, a
mantle extension around the edge of the foot. The epipodium varies in its
structural detail, coloration, and texture among species and is one way to
distinguish between the different species of abalone (Anderson 2003).

Two species of abalone have been introduced to Chile for culture purposes, the
Pacific abalone Haliotis discus hannai Ino 1953 and the Californian red abalone,
H. rufescens Swainson 1822. These two species are easily differentiated based on
their shell and epipodium morphology. The shell of the Pacific abalone is
normally green and variably rugose ru·gose or ru·gous
adj.
Having many wrinkles or creases; ridged or wrinkled.

rugose

marked by ridges; wrinkled. with brown and white epipodia. In contrast, the shell
of the California abalone cultured in Chile has a finely lined shell with variable
coverage of red and turquoise color and black, smooth epipodia.

Ten individuals obtained from the Center for Abalone Production of the
Universidad Catolica del Norte (CAP-UCN) in 2004 had the typical shell and
epipodial morphology of H. rufescens, but the coloration of the epipodia were
similar to that of H. discus hannai. Two possible hypotheses were considered: (1)
hybridization had occurred between Pacific and Californian abalone and (2)
phenotypic variation within H. rufescens caused the unusual coloration of the
epipodia. The latter might be explained by environmental adaptation and/or
genetic polymorphism.

Several authors have reported the existence of hybrids between abalone species
(Owen et al. 1971, Leighton & Lewis 1982, Brown 1995, Freeman 2001, Zhao et
al. 2004, Ibarra et al. 2005), and hybridization between H. rufescens and H. discus
hannai has been informally noted by commercial abalone growers in Chile (A.
Zuniga, pers. comm.). On the other hand, morphological variation produced by
genetic factors has been described for various bivalve bivalve, aquatic mollusk of
the class Pelecypoda ("hatchet-foot") or Bivalvia, with a laterally compressed
body and a shell consisting of two valves, or movable pieces, hinged by an elastic
ligament. molluscs, particularly with regard to shell color (Chanley 1961, Innes
& Haley 1977, Mitton 1977, Newkirk 1980, Adamkewicz & Castagna 1988,
Wada & Komaru 1990, Winkler Winkler may refer to:
• Winkler, Manitoba, a Canadian city
• Winkler (novel), by Giles Coren
• Winkler (crater), a crater on the Moon
• Winkler (surname), people with the surname Winkler or Winckler
See also
•
et al. 2001), abalones (Kobayashi et al. 2004) and other gastropods (Boulding &
Hay 1993, Rolan et al. 2004). Morphological variability produced by phenotypic
plasticity has also been reported for gastropod shells (Cole 1975, Wullschleger &
Jokela 2002, Veliz et al. 2001, Smith & Ruiz 2004). However, we have found no
published information on the variability of epipodial coloration within abalone
species.

The use of molecular markers has proved to be a useful tool for complex
taxonomic identification where morphological characteristics are ambiguous or
cryptic (e.g., Douek et al. 2002, Westheide et al. 2003, Miura et al. 2005, Park et
al. 2005). The randomly amplified polymorphic DNA-polymerase chain reaction
(RAPD-PCR) technique is a relatively simple and cheap method capable of
differentiating taxa taxa: see taxon. without the need to know their genomes
(Welsh & McClelland 1990, Williams et al. 1990). RAPDs are dominant markers
that result from the use of short (10 bases long) primers (synthetic
oligonucleotides) of random sequence that can amplify multiple segments of
genomic DNA by PCR. The number of segments depends on the number of sites
of the genome recognized by a particular primer. The main reason for the success
of RAPD analysis is the gain of a large number of genetic markers that require
small amounts of DNA without the need of a molecular characterization of the
genome of the taxa under study. Species in which such markers were used include
oyster genera Crassostrea, Saccostrea, and Striostrea (Klinbunga et al. 2000),
amphipods (Gammarus: Costa et al. 2004) and the tropical abalones Haliotis
asinina, H. ovina and H. varia var·i·a
n.
A miscellany, especially of literary works.

[Latin, from neuter pl. of varius, various.] (Klinbunga et al. 2004).

The goal of the present study was to obtain species-specific RAPD molecular
markers that could distinguish between H. discus hannai and H. rufescens and
verify if individuals with ambiguous morphology found at CAP-UCN represent
hybrids or intraspecific in·tra·spe·cif·ic also in·tra·spe·cies
adj.
Arising or occurring within a species: intraspecific competition. phenotypic
variability.

MATERIALS AND METHODS

A total of 27 individuals of each species (H. discus hannai and H. rufescens) were
obtained for this study from the CAP-UCN. About 2 [cm.sup.3] of muscle tissue
dissected from the foot of each sacrificed specimen was kept in a cryogenic tube
at -20[degrees]C until DNA was extracted. The 10 DNA samples of the abalones
with ambiguous morphology were obtained from the same tank of H. rufescens
hatchery hatchery

a commercial establishment dedicated to the hatching of bird eggs to provide day

old chicks and poults to the poultry industry.

hatchery liquid
the contents of unfertilized eggs. Used in petfood manufacture. broodstock (from
CAP-UCN) and were given to the Laboratory of Molecular Diversity when they
were already dead.

DNA extraction was carried out using a QIAamp Mini Kit (Qiagen Inc.). DNA
extractions were verified using the PCR reaction with the HCO-LCO universal
primers (Folmer et al. 1994) for the Cytochrome Oxidase cytochrome oxidase
n.
An oxidizing enzyme containing iron and a porphyrin, found in mitochondria and
important in cell respiration as an agent of electron transfer from certain
cytochrome molecules to oxygen molecules. I mitochondrial mitochondrial

pertaining to mitochondria.

mitochondrial RNAs
a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by
a mitochondrial-specific RNA polymerase, that account for about 4% of the total
cell RNA that gene. The presence and intensity of amplification (visualized in an
agarose agarose

more highly purified form of agar with similar uses to agar and widely used in the
separation of nucleic acid fragments. electrophoresis) was used as DNA quality
criterion. Clear and intense amplicons (bands) in the gel were considered to reflect
that the DNA used was of high quality and quantity. The extracted DNA was
stored at -20[degrees]C until further use.

To analyze a large number of individuals in each PCR reaction, two pools of DNA
were prepared for each species, each pool made up of equal aliquots of DNA from
eight different individuals of the species. Consequently, with two PCR reactions
per species, 16 individuals of each species would be screened. Each DNA pool
was assayed using 200 RAPD primers (RAPD primers 300-500, University of
British Columbia Locations
Vancouver
The Vancouver campus is located at Point Grey, a twenty-minute drive from
downtown Vancouver. It is near several beaches and has views of the North Shore
mountains. The 7. ) in independent reactions (each primer and DNA sample in an
independent reaction). The PCR reactions were carried out in a final volume of 10
uL, which contained 1X PCR buffer (200 mM Tris-HCL, pH 8.4, 500 mM KCL
KCL - Kyoto Common Lisp ), 1.5 mM Mg[Cl.sub.2] 0.1 mM of each dNTP, 0.2
[micro]M of each RAPD primer, 1.45 U Taq DNA polymerase DNA
polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes
catalyzing the template-directed incorporation of deoxyribonucleotides into a
DNA chain, particularly one using a DNA template.
..... Click the link for more information. (Invitrogen) and approximately 5 ng/
[micro]L of genomic DNA. In all the PCRs a negative and positive control were
included, to verify the absence of contamination in the samples and the success of
PCRs respectively.

Amplification was carried out in a PTC (PTC, Needham, MA, www.ptc.com)

Long a world leader in mechanical computer-aided design, manufacturing and
engineering software, PTC, through acquisitions and reorganization, has
transformed itself into a leading provider of Internet-based B2B solutions for
discrete manufacturers. 100-MJ Research thermal cycler, with an amplification
program starting with 2 min at 94[degrees]C, followed by 40 cycles consisting of
1 min at 94[degrees]C, 1 min at 42[degrees]C, and 2 min at 72[degrees]C,
followed by a final extension of 5 min at 72[degrees]C. The PCR products
(amplicons) were separated in a 6% denaturing Polyacrylamide gel
pol·y·a·cryl·a·mide gel
n.
A hydrated polymer consisting of a long chain of amide groups, used as a medium
for substances that undergo gel electrophoresis. containing 19:1 acrylamide
acrylamide /acryl·a·mide/ (ah-kril´ah-mid) a vinyl monomer used in the
production of polymers with many industrial and research uses; the monomeric
form is a neurotoxin. : bis-acrylamide and 5 M urea. Electrophoresis was
conducted using a SequiGen sequencing gel electrophoresis system (BIO-RAD
laboratories, Hercules, CA). Runs were performed at 6(>100 Watts and
55[degrees]C for 7-24 h depending on the sizes of the amplicons. Amplicons were
visualized on the gel using the Promega (Madison, WI) silver-staining protocol
(following manufacturer's instructions) and a 200 base pair (bp) DNA ladder as a
marker to estimate amplicon size. The primers that produced good amplification
(clearly defined bands that differentiated between the two abalone species) were
used in the next amplification step.

We screened the primers that simultaneously showed different and consistent

amplification results between species. The potentially useful primers were used to
amplify by PCR the DNA of each of the 27 individuals of H. discus hannai and H.
rufescens (i.e., primers were used to open the DNA pools). At this stage, primers
were considered as informative and reliable for identification and differentiation
between H. discus hannai and H. rufescens when they produced a specific
amplicon in all (27) individuals of one species and none from the other species.
These markers were then used to identify the specific status of the 10 abalone with
ambiguous morphology found at the CAP-UCN.

RESULTS

Of the 200 primers assayed with the pools of DNA from both species, 40 were
potentially useful for differentiating between the two abalone species. Four of the
40 primers found produced amplicons exclusive for H. discus hannai and 31 for
H. rufescens; whereas five primers simultaneously produced different size
amplicons unique for each species. The five primers that simultaneously produced
amplicons of different sizes and four other primers that produced good band
intensities, difference in molecular weight between amplicons of the two species,
and repeatability among assays were chosen and tested in 27 individuals of each
species.

All primers that produced amplicons of different sizes in both species showed
intraspecific polymorphisms when they were tested with the 27 individuals of
each abalone species, disallowing unequivocal differentiation between the two
species. However, two primers produced specific amplicons on the 27 individuals;
primer 356 (5' GCG GCG Genetics Computer Group
GCG Glucagon
GCG Good Corporate Governance
GCG Global Consumer Group
GCG Global Church of God
GCG Generalized Conjugate Gradient
GCG Global Change Game
GCG Geological Curators' Group
GCG Giant-Cell Granuloma GCC GCC: see Gulf Cooperation Council.

(compiler, programming) GCC - The GNU Compiler Collection, which currently

contains front ends for C, C++, Objective-C, Fortran, Java, and Ada, as well as
libraries for these languages (libstdc++, libgcj, etc). CTC CTC - Cornell Theory
Center T 3') for H. discus hannai and primer 368 (5' ACT TGT TGT Target
TGT Ticket Granting Ticket (Windows 2000 Kerberos security)
TGT Target Corp (stock symbol)
TGT Turbine Gas Temperature
TGT TDRSS Ground Terminal
TGT Tank Gunnery Trainer
TGT Target Tracker GCG G 3') for H. rufescens.

The PCR banding pattern (fingerprint) obtained with primer 368 shows an
amplicon of approximately 700 bp that is present in most of the individuals of H.
discus hannai, and in some of H. rufescens (Fig. 1, band A). Even though it was
not a specific amplicon for either of the two species, it was much more frequent
and intense in H. discus hannai. With this same primer, four anaplicons specific
for H. rufescens were found. These were of approximately 750 bp, 850 bp, 860
bp, and 1,190 bp, which are present in all individuals of H. rufescens, and in none
of H. discus hannai (Fig. 1, bands B-E), therefore making unequivocal
identification of the two species possible using primer 368. The banding patterns
obtained from assaying the DNAs of the 10 individuals with ambiguous
morphology with primer 368 showed the amplicons of approximately 750 bp and
850 bp unique to H rufescens, in some of the 10 individuals. The 750 bp amplicon
is only present in two specimens with ambiguous morphology and the 850 bp
amplicon in three individuals (Fig. 1).

The banding pattern produced with primer 356 shows an amplicon of

approximately 990 bp present in the 27 individuals of H. rufescens and in nine of
H. discus hannai (Fig. 2, band A). This amplicon, therefore, was not specific for
either of the two species, although it was more frequent and intense in H.
rufescens. The amplicons of approximately 450 bp and 460 bp, in contrast, were
present in all the individuals of H. discus hannai and none of the H. rufescens
(Fig. 2, bands B and C). Consequently, these two amplicons allow unequivocal
species identification. The RAPD banding pattern of the 10 individuals with
ambiguous morphology with primer 356 did not show discriminatory amplicons
unique to H. discus hannai, whereas all the individuals with ambiguous
morphology showed the 990 bp amplicon, frequent and more intense in H.
rufescens.

[FIGURE 1 OMITTED]

DISCUSSION

Our results strongly suggest that the 10 specimens with ambiguous morphology
found at the CAP-UCN correspond to H. rufescens and that there is phenotypic
variability within this species. The banding patterns from the RAPD-PCRs carried
out on the DNA of these specimens show greater similarities with the banding
patterns of H. rufescens. Some of the DNA of the individuals with ambiguous
morphology show amplicons unique to H. rufescens (750 bp and 850 bp with
primer 368), whereas the amplicons unique to H. discus hannai (450 bp and 460
bp with primer 356) were not detected in any of them. The specific amplicons of
both species would be expected to appear in the banding pattern of the individuals
with ambiguous morphology if the specimens in question had been hybrids.
Although the fingerprint is not always consistent, the whole fingerprint of 10
specimens with ambiguous morphology is visibly more consistent with the
fingerprint of H. rufescens than H. discuss hannai (Fig. 1 and Fig. 2). The
inconsistency in the fingerprint of the specimens with ambiguous morphology
could be caused by the inadequate maintenance of tissue samples that produces
poor and degraded DNA. These results emphasize the importance of good
maintenance of tissues that will be used for molecular work. Unfortunately we did
not have more or fresh tissues of specimens with ambiguous morphology to
perform the study.

[FIGURE 2 OMITTED]

The results show that RAPD-PCR is a simple and efficient method to identify and
differentiate between H. discus hannai and H. rufescens. The banding patterns
obtained from some RAPDPCR show specific amplicons for molecular
differentiation.

The RAPD markers tested herein, allowed us to reject the hypothesis that the 10
individuals with ambiguous morphology were hybrids between the two species of
abalone. The banding patterns produced with the DNA of the 10 individuals with
ambiguous morphology did not simultaneously carry the amplicons obtained with
primers 368 and 356, for H. rufescens and H. discus hannai respectively. Other
molecular genetic tools may allow better discrimination of hybrids and could be
used to corroborate To support or enhance the believability of a fact or assertion
by the presentation of additional information that confirms the truthfulness of the
item.

The testimony of a witness is corroborated if subsequent evidence, such as a

coroner's report or the testimony of other our findings, including microsatellites
(Ibarra et al. 2005), sequencing (Spies et al. 2006) and allozymes (Brown 1995,
Ibarra et al. 2005).

Variability in the size and morphology of gastropod shells can be influenced both
genetically and environmentally (Boulding & Hay 1993, Trussell & Etter 2001,
Jordanes et al. 2003, Rolan et al. 2004). Phenotypic plasticity represents the
potential of an organism to alter its phenotype caused by environmental stimuli
(Levins 1968, Adler & Harvell 1990). Genetic polymorphism is caused by the
presence of two or more alleles at a given locus and depending on the allele
allele (əlēl`): see genetics.
allele

Any one of two or more alternative forms of a gene that may occur alternatively at
a given site on a chromosome. combination, can be expressed as different
phenotypes. Because the 10 abalone individuals showing ambiguous morphology
were obtained from a single breeding population of H. rufescens (even though
their parentage PARENTAGE. Kindred. Vide 2 Bouv. Inst. n. 1955; Branch;
Line. is unknown) and cultured under the same hatchery conditions (i.e., same
food and environmental conditions) it seems improbable that the morphological
variability found was a manifestation of phenotypic plasticity, and is more likely
ascribed to genetic polymorphism. The present study represents the first published
report on intraspecific variability in the color of the epipodium of H. rufescens (or
any other abalone species). Other experiments therefore could be carried out in
which crosses are made between abalones to confirm the hypothesis that the color
variation represents intraspecific polymorphism (if more individuals with the trait
are found). The crosses would allow verification of the stability of a character
throughout the ontogenetic on·to·ge·net·ic
adj.
Of or relating to ontogeny. development and possible patterns of character
segregation. Additionally, molecular markers associated with the trait could be
developed.

LITERATURE CITED

Adamkewicz, L. & M. Castagna. 1988. Genetics of shell color and pattern in the
bay scallop scallop or pecten, marine bivalve mollusk. Like its close relative the
oyster, the scallop has no siphons, the mantle being completely open, but it differs
from other mollusks in that both mantle edges have a row of steely blue "eyes"
and Argopeeten irradians. J. Hered. 79:14-17.

Adler, F. & C. D. Harvell. 1990. Inducible defences, phenotypic variability and

biotic biotic /bi·ot·ic/ (bi-ot´ik)
1. pertaining to life or living matter.

2. pertaining to the biota.

bi·ot·ic
adj.
1. Relating to life or living organisms. environments. Trends Ecol. Evol. 5:407-
410.

Anderson, G. 2003. Marine Science. Chapter 6.2.1 at http://www.

biosbcc.net/ocean/marinesci/06future/abintro.htm

Brown, L. D. 1995. Genetic evidence for hybridization between Haliotis rubra and
H. laevigata. Mar. Biol. 123:89-93.

Boulding, E. G. & T. K. Hay. 1993. Quantitative genetics of shell form of an

intertidal in·ter·tid·al
adj.
Of or being the region between the high tide mark and the low tide mark.

in snail: constrains on short-term response to selection. Evol. 47:576-592.

Chanley, P. E. 1961. Inheritance of shell markings and growth in the hard clam
Venus mercenaria. Proc. Natl. Shellf. Assoc. 50:163-169.

Cole, T. J. 1975. Inheritance of juvenile shell colour of the oyster drill, Urosalpix
cinerea. Nature 257:794-795.
Costa, F. O., M. R. Cunha, T. Neuparth, C. W. Theodorakis, M. H. Costa & L. R.
Shugart. 2004. Application of RAPD DNA fingerprinting DNA fingerprinting or
DNA profiling, any of several similar techniques for analyzing and comparing
DNA from separate sources, used especially in law enforcement to identify
suspects from hair, blood, semen, or other biological materials found at in
taxonomic identification of amphipods: a case-study with Gammarus species
(Crustacea: Amphipoda). J. Mar. Biol. Assoc. UK. 84:171-178.

Douek, J., I. Barki, D. Gateno & B. Rinkevich. 2002. Possible cryptic speciation
speciation

Formation of new and distinct species, whereby a single evolutionary line splits
into two or more genetically independent ones. One of the fundamental processes
of evolution, speciation may occur in many ways. within the sea anemone Actinia
equina complex detected by AFLP markers. Zool. J. Lin. Soc. 136:315-320.

Folmer, O., M. Black, R. Hoeh, R. A. Lutz & R. Vrijenhoek. 1994. DNA primers
for amplification of mitochondrial cytochrome oxidase subunit I from diverse
metazoan metazoan

member of the zoological division of Metazoa. invertebrates. Mol. Mar. Biol.

Biot. 3:294-299.

Freeman, K. A. 2001. Aquaculture aquaculture, the raising and harvesting of

fresh- and saltwater plants and animals. The most economically important form of
aquaculture is fish farming, an industry that accounts for an ever increasing share
of world fisheries production. and related biological attributes of abalone species
in Australia- a review. Fish. Res. Rep. West. Aust. 128:1-48.

Ibarra, A. M., N. K. Hernandez-Ibarra, P. Cruz, R. Perez-Enriquez, S. Avila & J.

L. Ramirez. 2005. Genetic certification of presumed hybrids of blue and red
abalone (Haliotis fulgens Philippi and H. rufescens Swainson). Aquac. Res.
36:1356-1368.

Innes, D. J. & L. E. Haley. 1977. Inheritance of a shell-color polymorphism in the

mussel mussel, edible freshwater or marine bivalve mollusk. Mussels are able to
move slowly by means of the muscular foot. They feed and breathe by filtering
water through extensible tubes called siphons; a large mussel filters 10 gal (38
liters) of water per day. . J. Hered. 68:203-204.

Jordanes, K., P. Van Riel ri·el

n.
See Table at currency.

[Origin unknown.]
Noun 1. riel - the basic unit of money in Cambodia; equal to 100 sen & T.
Backeljau. 2003. Molecular and morphological discrimination between the
pulmonate pul·mo·nate
adj.
1. Having lungs or lunglike organs.

2. Of or belonging to the Pulmonata, a subclass of gastropods including terrestrial

snails and slugs and certain freshwater snails that are capable of breathing air
through land snail Zonitoides nitidus and Z. excavatus. J. Moll. Stud. 69: 295-
300.

Klinbunga, S., P. Ampayup, A. Tassanakajon, P. Jarayabhand & W. Yoosukh.

2000. Development of species-specific markers of the tropical oyster (Crassostrea
belcheri) in Thailand. Mar. Biotechnol. 2:476-84.

Klinbunga, S., P. Ampayup, R. Leelatanawit, A. Tassanakajon, I. Hirono, T. Auki,

P. Jarayabhand & P. Menasveta. 2004. Species identification of the tropical
abalone (Haliotis asinina, Haliotis ovina and Haliotis varia) in Thailand using
RAPD and SCAR markers. J. Bioch. Mol. Biol. 37:213-222.

Kobayashi, T., I. Kawahara, O. Hasekura & A. Kijima. 2004. Genetic control of

bluish blu·ish also blue·ish
adj.
Somewhat blue.

blu ish·ness n. shell color variation in the Pacific abalone, Haliotis discus hannai.
J. Shellfish Res. 23:1153-1156.

Leighton, D. L. & C. A. Lewis. 1982. Experimental hybridization in abalone, hit.

J. Invertebr. Repr. 5:273-82.

Levins, R. 1968. Evolution in changing environments: some theoretical

explorations. Princeton, N J: Princeton Univ. Press, 132 pp.

Mitton, J. B. 1977. Shell color and pattern variation in Mytilus edulis and its
adaptive significance. Chesapeake Sci. 18:387-390.

Miura, O., A. M. Kuris, M. E. Torchin, R. F. Hechinger, E. J. Dunham & S.

Chiba. 2005. Molecular--genetic analyses reveal cryptic species of trematodes in
the intertidal gastropod Batillaria cumingi (Crosse). Int. J. Parasit. 35:793-801.

Newkirk, G. F. 1980. Genetics of the shell color in Mytilus edulis L. and the
association of growth rate with shell color. J. Exp. Mar. Biol. Ecol. 47:89-94.

Owen, B., J. H. McLean & R. J. Meyer. 1971. Hybridization in the Eastern Pacific
abalone (Haliotis). Sci. Bull. Nat. Hist. Mus. Los Ang. City. 9:1-37.
Park, K., J. K. Park, J. Lee & K. S. Choi. 2005. Use of molecular markers for
species identification of Korean Perkinsus sp isolated from Manila clams
Ruditapes philippinarum. Dis. Aquat. Org. 66: 255-263.

Rolan, E., J. Guerra-Varela, I. Colson, R. N. Hughes & E. Rolan-Alvarez. 2004.

Morphological and genetic analysis of two sympatric sym·pat·ric
adj. Ecology
Occupying the same or overlapping geographic areas without interbreeding. Used
of populations of closely related species. morphs of the dogwhelk Nucella lapillus
la·pil·lus
n. pl. la·pil·li
A small, solidified fragment of lava.

[Latin, diminutive of lapis, stone. (Gastropoda: Muricidae) from Galicia

(northwestern Spain). J. Moll. Stud. 70:179-185.

Smith, N. F. & G. M. Ruiz. 2004. Phenotypic plasticity in the life history of the
mangrove mangrove, large tropical evergreen tree, genus Rhizophora, that grows
on muddy tidal flats and along protected ocean shorelines. Mangroves are most
abundant in tropical Asia, Africa, and the islands of the SW Pacific. snail
Cerithidea scalariformis. Mar. Ecol. Prog. Ser. 284:195-209.

Spies, I. B., S. Gaichas, D. E. Stevenson D. E. Stevenson (1892-1973) - married

name Dorothy Emily Peploe - was a Scottish author of light romantic novels. Her
father was the lighthouse engineer David Alan Stevenson and she was distantly
related to the writer Robert Louis Stevenson. , J. W. Orr & M. F. Canino. 2006.
DNA-based identification of Alaska skates (Amblyraja, Bathyraja and Raja:
Rajidae) using cytochrome c oxidase The enzyme cytochrome c oxidase or
Complex IV (PDB 2OCC, EC 1.9.3.1) is a large transmembrane protein complex
found in bacteria and the mitochondrion. Function
It is the last protein in the electron transport chain.
..... Click the link for more information. submit 1 (coI) variation. J. Fish Biol.
69:283-292.

Trussell, G. C. & R. J. Etter. 2001. Integrating genetic environmental forces that

shape the evolution of geographic variation in a marine snail. Genetica 112-
113:321-337.

Veliz, D., C. Guisado & F. M. Winkler. 2001. Morphological, reproductive, and

genetic variability among three populations of Crucibulum quiriquinae
(Gastropoda: Calyptraeidae) in northern Chile. Mar. Biol. 139:527-534.

Wada, K. T. & A. Komaru. 1990. Inheritance of white coloration of the prismatic

pris·mat·ic also pris·mat·i·cal
adj.
1. Of, relating to, resembling, or being a prism.
2. Formed by refraction of light through a prism. Used of a spectrum of light.

3. Brilliantly colored; iridescent. layer of shells in the Japanese pearl oyster

Pincada fucata martensii and its importance in the pearl culture industry. Nippon
Suisan Gakkaishi 56:1787-1790.

Welsh, J. & M. McClelland. 1990. Fingerprinting genomes using PCR with

arbitrary primers. Nucleic Acids Nucleic acids
The cellular molecules DNA and RNA that act as coded instructions for the
production of proteins and are copied for transmission of inherited traits. Res.
19:5275-5279.

Whestheide, W. & H. Schmidt. 2003. Cosmopolitan versus cryptic meiofaunal

Polychaete polychaete

Any of about 5,400 species of marine worms of the annelid class Polychaeta,
having a segmented body with many setae (bristles) on each segment. Species,
often brightly coloured, range from less than 1 in. (2.5 cm) to about 10 ft (3 m)
long. species: An approach to a molecular taxononly. Helg. Mar. Res. 57:1-6.

Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rafalski & S. V. Tingey.

1990. DNA polymorphism DNA polymorphism
n.
A condition in which one of two different but normal nucleotide sequences can
exist at a particular site in a DNA molecule. amplified by arbitrary primer are
useful as genetic markers. Nucleic Acids Res. 18:6531-6535.

Winkler, F. M., B. F. Estevez, L. B. Jollan & J. P. Garrido. 2001. Inheritance of

the general shell color in the scallop Argopecten purpuratus (Bivalvia:
Pectinidae). J. Hered. 92:521-525.

Wullschleger, E. B. & J. Jokela. 2002. Morphological plasticity and divergence in

life-history traits between two closely related freshwater snails, Lymnaea ovata
and Lymnaea peregra. J. Moll. Stud. 68:1-5.

Zhao, H., J. Zhang, L. Huang & L. Sun. 2004. Biological zero point in hybrid
Pacific abalone. J. Shellfish Res. 23:1113-1116.

S. A. MARIN, (1) P. A. HAYE, (1,2) * S. MARCHANT (1) AND F. M.

WINKLER (1,2)

(1) Departamento de Biologia Marina, Facultad de Ciencias del Mar, Universidad

Catolica del Norte; (2) Centro de Estudios Avanzados en Zonas Aridas (CEAZA),
Larrondo 1281. Coquimbo, Chile

* Corresponding author. E-mail: phaye@ucn.cl