Marine Biology (2001) 139: 527±534 DOI 10.1007/s002270100601
D. VeÂliz á C. Guisado á F.M. Winkler
Morphological, reproductive, and genetic variability among three populations of Crucibulum quiriquinae (Gastropoda: Calyptraeidae) in northern Chile
Received: 3 November 2000 / Accepted: 5 April 2001 / Published online: 9 June 2001 Ó Springer-Verlag 2001
Abstract Crucibulum quiriquinae (Lesson, 1830) is the only species of Crucibulum currently recognized in northern Chile. Recent analysis of three Crucibulum populations obtained in northern Chile demonstrates the existence of morphological, genetic, and reproductive dierences among populations. Two populations present in BahõÂ a Tongoy (30°15¢S), one inhabiting the shells of the snail Turritella cingulata and the other shells of the pectinid Argopecten purpuratus, showed morphological dierences. However, both had planktonic larval development and show low genetic divergence (D=0.002). A third population from BahõÂ a La Herradura (29°58¢S), which also inhabits the shells of T. cingulata, did not show morphological dierences compared with its counterpart from BahõÂ a Tongoy. However the BahõÂ a La Herradura population had intracapsular development and metamorphosis, and a larger genetic distance (D=0.06) from both Tongoy populations. The results of the reproductive and genetic analyses strongly suggested that the two Tongoy populations, although showing morphological dierences, are biologically the same species, Crucibulum quiriquinae, whereas the La Herradura population is a new species.
Communicated by P.W. Sammarco, Chauvin D. VeÂliz á C. Guisado á F.M. Winkler (&) Departamento de BiologõÂ a Marina, Universidad CatoÂlica del Norte, Sede Coquimbo, Casilla 117, Coquimbo, Chile E-mail:
[email protected] C. Guisado Instituto de Oceanologõ a, Facultad de Ciencias del Mar, Universidad de Valparaõ so, Casilla 13-D, VinÄa del Mar, Chile Present address: D. VeÂliz GIROQ, DeÂpartement de Biologie, Universite Laval, QueÂbec, Canada G1K 7P4
Introduction Criteria used in the taxonomy of species in the genus Crucibulum, a marine gastropod, have varied historically with the investigators who studied them. Broderip (1835) assigned a high speci®c diversity to this genus in the American Paci®c. By the turn of the century, Dall (1909) recognized only a few species. Recent authors, such as Keen (1971), remain conservative in their estimates of the number of species and recognize diculties in species identi®cation primarily related to the simplicity of the shells and the morphological variability produced by their sessile habit. It is thus necessary to examine characters that are not as strongly in¯uenced by environmental factors as shell morphology to distinguish or identify species. A conical shell with a subcentral apex and an internal cup-shaped septum morphologically characterizes the genus Crucibulum (Keen 1971). The only species of this genus reported for northern Chile is Crucibulum quiriquinae Lesson, 1830 (Marincovich 1973). Our preliminary observations in subtidal habitats of the Chilean coast detected three populations of Crucibulum that appeared to show disparity in morphology and reproductive strategies. Two of the populations were observed in BahõÂ a Tongoy (30°15¢S), one inhabiting the surface of the snail Turritella cingulata Sowerby, 1825, and the other the shells of the scallop Argopecten purpuratus Lamarck, 1822. Both Tongoy populations dier markedly in shell morphology but have similar larval development. A third population occurs in BahõÂ a La Herradura (29°58¢S), also living on T. cingulata. Specimens from this population have similar shell morphology to their counterparts in BahõÂ a Tongoy about 30 km to the south but appeared to dier from them in larval development. The present study characterizes the morphologies of these Crucibulum populations and incorporates aspects of their intracapsular development and allozymic variability in an attempt to de®ne their taxonomic status.
528
Materials and methods Crucibulum specimens were obtained from three subtidal populations in the Coquimbo Region of Chile, between August 1996 and January 1997. Several samples from each population were collected within this period. Two populations were sampled from BahõÂ a Tongoy (30°16¢S, 71°37¢W; Fig. 1). Specimens of the ®rst population (population I) were recovered individually or in very low numbers from the edge of the upper valves of the scallop Argopecten purpuratus, close to the ®shermen's pier at Puerto Aldea from a depth of 6±8 m. The second population (population II) occurred o ``La Puntilla'' about 2 km northeast of the ®rst population, also collected from 6±10 m. One or very few specimens were found attached to the body whorls of individual Turritella cingulata (Gastropoda: Turritellidae). Specimens of the third population (population III) were collected from BahõÂ a La Herradura (29°58¢S, 71°21¢W; Fig. 1), at a depth of 4±6 m, where they also live on the shells of T. cingulata. In BahõÂ a La Herradura no Crucibulum
Fig. 1 Crucibulum: Sampling sites on the northern Chilean coast, showing details of BahõÂ a Tongoy and BahõÂ a La Herradura. Roman numerals I, II, and III indicate the collection sites of each population
Table 1 Crucibulum: Enzyme systems, tissues, and buers used in genetic analysis of three Crucibulum populations from the Coquimbo Region, Chile. ECN Enzyme Commission number
specimens were found living on A. purpuratus shells. All specimens were hand collected with SCUBA diving, and the egg masses brooded by each individual were also collected. Specimens were maintained separately in appropriately labeled plastic bags at ±20°C for subsequent morphological and genetic analyses. Egg masses were maintained separately and ®xed in 3% glutaraldehyde in 1 lm ®ltered seawater. Morphological analyses include description of general shell morphology and external shell ornamentation, form of the aperture, position of the apex, form of the internal septum, internal coloration of the shell, range of sizes of each of the sexual phases, and morphological analysis of the radula. The sexual phases, including juveniles, males, intersexes, and females, were determined on the basis of penis size and color of the gonad, using methods of Hendler and Franz (1971). Radulae were obtained by dissection and washed in 5% NaCl in distilled water for 5 min to remove extraneous tissue and organic matter. They were then oven dried at 40°C for 15 min, sputter coated with gold in a JEOL JFC-1100 ``Fine Coat'' apparatus, and then observed and photographed using a JEOL T300 scanning electron microscope. A minimum of 30 individuals from each population were measured, and the number of egg capsules present and the general morphology ofthe egg capsules were noted. The type of intracapsular development was characterized on the basis of developmental stage that emerged from the capsule (veliger larvae or juveniles), as well as the type of nutrition occurring during intracapsular development. Egg size and number per capsule were determined in 14, 6, and 18 females from populations I, II, and III, respectively. Five to ten capsules per brood were recorded. The number of veliger larvae and juveniles per brood was estimated, using no less than 30 measurements for each population. Observations (qualitative and quantitative) were made using a Nikon Biophot light microscope equipped with an ocular micrometer. Statistical dierences between the sizes and numbers of eggs per brood were tested between populations using one-way ANOVA with the a posteriori Tukey's test (Sokal and Rholf 1969; Steel and Torrie 1988). Genetic variation was assessed using 10% starch gel electrophoresis. Samples of muscle and gonad/digestive gland from each population were placed individually in 1.5 ml Eppendorf tubes with an equal volume of buer solution (EDTA 2Na 0.001 M, Tris 0.01 M, pH=6.8) and homogenized. Electrophoresis was carried out following methods given by Aebersold et al. (1987); buer systems and tissues utilized are listed in Table 1. Nomenclature of loci and alleles followed the general recommendations proposed by Shaklee et al. (1990) for ®shes.
Enzyme
Abbreviation
ECN
Tissue
Buera
Aspartate aminotransferase Acid phosphatase Adenylate kinase Creatine kinase Malic enzyme Esterase Esterase D Glucose 6 phosphate isomerase Hexokinase L-iditol dehydrogenase Isocitrate dehydrogenase Malate dehydrogenase Mannose-6-phosphate isomerase Peptidase B General proteins Phosphoglucomutase Superoxide dismutase
AAT ACP AK CK EM EST EST-D GPI HK IDDH IDH MDH MPI PEP-B PG PGM SOD
2.6.1.1 3.1.3.2 2.7.4.3 2.7.3.2 1.1.1.40 3.1.1.± 3.1.±.± 5.3.1.9 2.7.1.1 1.1.1.14 1.1.1.42 1.1.1.37 5.3.1.8 3.4.±.±
Muscle Gland-gonad Muscle Muscle Muscle Gland-gonad Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle Muscle
TBE TBCL TBCL ACE 7.0 ACE 7.0 TBCLE ACE 7.0 ACE 6.5 TBE TBCL ACE 7.0 ACE 6.5 TBCL TBE TBE TBE TBCL
a
5.4.2.2 1.15.1.1
Buer systems used: ACE pH 6.5 and 7.0, Clayton and Tretiak (1972); TBCL pH 8.0/8.7, Ridway et al. (1970); TBCLE pH 8.0/8.7, Ridway et al. (1970)+EDTA; TBE pH 8.7, Boyer et al. (1963)
529 The homogeneity of allele frequencies between populations was tested using a v2 test for allelic frequency homogeneity (Nei 1987). The genetic identity (I) and standard genetic distance (D) between populations were estimated according to Nei (1972, 1978).
Results Morphological characteristics The only morphological characteristics shared in common among the three populations were a convex external shell with a subcentral apex bent to the right, and the number and position of cusps on the radula teeth (Table 2). The population from Puerto Aldea living on scallop shells (population I) showed marked morphological dierences from the others, having a circular aperture and external sculpture, often in the form of marked ridges. Separation of the internal septum from the external shell in the basal region occurred in intersex and female individuals (Fig. 2a, b). This population also had the greatest range in size. Populations living on Turritella shells (populations II, III) had very similar morphological and morphometric characteristics, including complete union of the shell and the internal septum on the left side of the shell in all sexual stages. These populations diered only in internal coloration of the shell, which is white in the Puerto Aldea population (population II) and brown in the La Herradura population (population III) (Fig. 2c, d).
Intracapsular development The ovoid egg capsules with laminar peduncles had the same morphology among all three populations (Table 3). In populations I and II all the eggs within capsules cleaved and produced swimming veliger larvae with relatively large velar lobes (Fig. 3A, B). Planktotrophic veligers hatch from the capsules. Population I had larger eggs than those of population II (F=31.77; n=385; P
