Testing the accuracy of morphological identification of northern quahog larvae.
Morphology (Animals) (Research)
Quahogs (Physiological aspects)
Perino, Laurie L.
Padilla, Dianna K.
Doall, Michael H.
|Publication:||Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2008 National Shellfisheries Association, Inc. ISSN: 0730-8000|
|Issue:||Date: Dec, 2008 Source Volume: 27 Source Issue: 5|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: United States Geographic Code: 1USA United States|
ABSTRACT Bivalve larvae in mixed samples collected from the field
have traditionally been identified through morphological differences
among species. It is difficult, however, to use this method accurately
because of overlapping size ranges and similar shapes of the larvae of
many species. We used the molecular technique developed by Hare et al.
(2000) to test the accuracy of morphological identification of
Mercenaria mercenaria (L.) larvae from plankton samples collected from
the Great South Bay and Coecles Harbor on Long Island, New York. We
found that morphology is unreliable as the only means of identification
for bivalve larvae in a mixed field sample, and a very high false
positive rate of identification of M. mercenaria (100% of 71 larvae were
misidentified). Morphological characteristics may be used to eliminate
larvae from a field plankton sample, as the false negative rate for M.
mercenaria was only 1.4% (n = 140). To determine larval bivalve
densities accurately, other techniques in addition to those based on
morphological characteristics, such as the molecular technique used in
this study, must be used.
KEY WORDS: Mercenaria, quahog, hard clam, larvae, identification
The northern quahog, also known as the hard clam, Mercenaria mercenaria (L.), is an ecologically and economically important clam. Functionally, it is a suspension feeder with the ability to alter its environment when in high densities (Cohen et al. 1984, Nichols 1985, Alpine & Cloern 1992, Cerrato et al. 2004). Hard clams can affect the growth of other organisms through direct competition for phytoplankton resources with other suspension feeders and by reducing phytoplankton abundance and increasing light penetration, thereby promoting growth of plants and phytoplankton. Hard clams may be able to control the growth of some harmful algae (Cerrato et al. 2004), and may be able to affect the overall resistance of the system to a disturbance, including harmful algal blooms. Hard clams are also an important part of local economies, especially along the east coast of the United States, where it is commercially harvested. The bays around Long Island, New York historically accounted for about half of the United States landings of hard clams (Schubel 1991). In the mid 1970s and early 1980s, large harvests of a fairly unregulated fishery led to the precipitous decline of clam populations. Although fishing has been reduced, to date there has been no recovery of local populations (personal communications from the National Marine Fisheries Service, Fisheries Statistics Division, Silver Spring, MD). Low fertilization rates may inhibit recovery because the clams are in such low densities that their gametes are too dilute for successful fertilization, as has been suggested for other free spawning marine invertebrates (Pennington 1985, Levitan 1991).
With this in mind, The Nature Conservancy has recently established no harvest sanctuaries of adult clams in bays around Long Island. The goal of these sanctuaries is to re-establish a self-sustained population of clams and restore lost ecosystem function. The adult clams have been transplanted to locally high density patches in the hopes of improving fertilization success.
To assess the success of these sanctuaries, researchers need to know if the adults are developing gonads, if they spawn, if their gametes are successfully fertilized, and if the larvae are growing and surviving to metamorphosis. Determining fertilization success and the growth of larvae requires the examination of plankton samples collected from the field; therefore the accurate identification of larvae of bivalves in mixed plankton samples is essential.
Traditionally, morphological characteristics have been used to identify larvae of bivalves from field samples (Loosanoff et al. 1966, Chanley & Andrews 1971). The size, stage, and shape of the larval shell are used as species-specific identifiers; however, because different species may have overlapping size ranges and look very similar to one another, it is difficult to have confidence in the accuracy of such identifications. This is especially true for smaller larvae in the D-stage, the first shelled veliger stage. The use of fine structures of hinge teeth have also been proposed as a means of identifying bivalve larvae, but this can be a time-consuming procedure (Lutz et al. 1982).
We tested the accuracy of using morphological characteristics for identifying larvae of Mercenaria mercenaria in plankton samples collected from the field. We determined the error rates of false identifications (identifying a larva as M. mercenaria when they are in fact another species, as well as failing to identify correctly a larva as M. mercenaria) and used these rates to make recommendations for techniques used for future larval studies.
MATERIALS AND METHODS
Larval Abundance Determined from Shell Morphology
Abundances of bivalve larvae were estimated by concentrating the plankton samples collected from the field to 50 mL total volume. A 5 mL subsample for each plankton sample was examined through a dissecting microscope and all larvae of bivalves were counted. Cross-polarized light was used to identify larvae easily as shells appear to glow under this lighting, making larval bivalves stand out when among other plankton. At least two 5 mL subsamples were counted. If after the first two subsamples the total count of larval bivalves was not at least 100, additional subsamples were counted until either the count reached at least 100 or until a maximum of 6 subsamples were counted. Estimates of the total density of bivalve larvae were then calculated using the following formula:
[(Total number of larvae counted/Number of subsamples) x 10]/30 = Number of bivalve larvae/L
The abundance of larvae of Mercenaria mercenaria in plankton samples was estimated by using morphological characteristics as described by Loosanoff et al. (1966) and Chanley and Andrews (1971). They found that larvae of M. mercenaria grow isometrically, thus, the general shape of the larvae (length to width ratio) as well as a size range for length, width and hinge length (as described below) were used as our criteria for identifying larvae as M. mercenaria. Laboratory-cultured larvae of M. mercenaria were obtained by spawning adult hard clams in the laboratory. These larvae were collected and stored in 80% ethanol and the length and width of larvae of different ages was measured and then compared with the relationship determined by Loosanoff et al. (1966) for M. mercenaria.
[FIGURE 1 OMITTED]
Larvae from M. mercenaria spawned in the laboratory were measured to determine the low-end size cutoffs for morphological identification. From these measurements, we determined the size cutoffs for those larvae to be measured from plankton samples collected from the field. We determined the hinge length cutoff to be 67 [micro]m, width cutoff to be 80 [micro]m, and length cutoff to be 100 [micro]m. No high-end cutoff values were used, as all of the larvae in the plankton samples collected from the field were within the acceptable upper range for M. mercenaria. The developmental stage of the larva was also used as a guide for identification. At a length of approximately 125 [micro]m, larvae of M. mercenaria develop an umbo, so those larvae with an umbo at a smaller length were excluded. All larvae were measured with a computer assisted image analysis system (Image-Pro Plus, Media Cybernetics) and only those matching the earlier mentioned shape and size criteria were counted as M. mercenaria, and their density was estimated by using the formula shown earlier.
Molecular Verification of Larval Identity
A species-specific multiplex PCR protocol developed by Hare et al. (2000) was used to test for false positive (identifying a larva as M. mercenaria when it is not) and false negative (identifying a larva as not M. mercenaria when it is) identification rates when using morphology (n = 211). An individual larva from a field sample was rinsed in distilled water to remove all alcohol present from preservation and the length, width, and hinge length of the larva was measured and used to determine whether each larva fit the morphological criteria for M. mercenaria. Each larva was also staged (D-stage or umbonal) to determine if its size was within the range for a M. mercenaria at the same stage. Individual larvae were then transferred to labeled PCR tubes so that each larva could be tracked through the PCR process.
Species-specific primers that result in a diagnostic DNA fragment length were used to test for species identification as M. mercenaria. To get the specificity needed from the primers, fragments from mitochondrial cytochrome oxidase I were targeted. Qiagen HotStar Master Mix was used for PCR and the samples were run in a thermocycler for an initial period of 15 min to extract the DNA and to activate the taq mixture. The PCR product was then run on an agarose gel stained with SYBR safe dye to illuminate the DNA fragments. For every plankton sample that was tested, a control of a known larva of M. mercenaria (from larvae of M. mercenaria produced in laboratory spawnings) and a blank control (no larva present) were included. Only those runs that produced a band for the hard clam control and no band for the blank control were used.
For six samples in Great South Bay and four samples in Coecles Harbor, a random subset of the larvae measured for each sample was also identified using the molecular technique. We used the percent of M. mercenaria found using the molecular technique to estimate the density of M. mercenaria in each of the samples. This number was then compared with estimates of larval densities obtained when using the morphological technique as a measure of the accuracy of morphological identification.
We found a significant relationship between larval shell width and length for known M. mercenaria larvae ([r.sup.2] = 0.83, Fig. 2), with a slope of 1, indicating isometric growth. There was, however, substantial scatter around the regression line. This relationship between larval shell length and width was similar to that found by Loosanoff et al. (1966), who concluded that larvae grow isometrically.
Larvae from 10 different Coecles Harbor and Great South Bay samples were used to test the accuracy of morphological identification by using molecular identification. For the majority of samples, morphological identification resulted in a large overestimate of the relative and absolute abundances of M. mercenaria larvae in the plankton (Fig. 3, Table 1). For example, in the June 30th sample from Coecles Harbor, no larvae of M. mercenaria were found with molecular identification, but based on morphology alone over 60% of the larvae in the sample were identified as M. mercenaria.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Error rates for morphological identification were tested using a subset of 211 larvae from 10 samples collected from Coecles Harbor and Great South Bay. The false positive rate for morphological identification was 100%. Of the 71 larvae identified as M. mercenaria, based solely on morphology, none were M. mercenaria based on molecular identification (Table 2). The false negative rate of larval identification based on morphology alone was only 1.4% of 140 individuals tested (Table 2).
In his classic study (and currently the only other field study of larval abundance of M. mercenaria in Long Island waters) Carriker (1961) used the relatively constant length to width ratio of larvae to quantify M. mercenaria larvae in plankton samples collected from the field. It is clear now that morphological shell characteristics are a very unreliable means of identifying larvae of M. mercenaria in Long Island waters. None of the larvae tested that were identified as M. mercenaria on the basis of morphology were in fact M. mercenaria based on a molecular identification. Morphological identification resulted in a false positive rate of 100%. This means that all of the larvae identified as M. mercenaria were incorrectly identified and were, in fact, some other, unknown bivalve species. Thus any estimates of larval densities based solely on morphological criteria should be reassessed. Morphology instead may be a useful tool for excluding larvae from being identified as M. mercenaria. The false negative rate was low, thus very few larvae that were identified as being another species were in fact M. mercenaria. Although it is not useful in correctly identifying larvae as being M. mercenaria, morphological identification can be used in mixed plankton samples to exclude certain bivalve larvae as being M. mercenaria. This could prove useful when there are hundreds of larvae per liter in a sample collected from the field. The molecular technique used here can then be used on a selected subsample of larvae to reduce the number of larvae needed to be processed to obtain an estimate of larval M. mercenaria density, therefore minimizing the cost of supplies for PCR.
The accuracy of density of larvae from field collected samples based on morphological identification of hard clam larvae varied by sample and differed by collection site (Fig. 3). The number of larvae produced by other species that are morphologically similar to M. mercenaria must vary among sites, and those other species must have different patterns in spawning, as mistaken larvae were not constantly found in high abundance. On some sample dates incorrectly identified larvae were very abundant (e.g., Coecles Harbor July 15th sample), whereas on others (e.g., Great South Bay July 14th sample) they were very rare.
The unreliability of morphology as a method of identification seems to result from the presence of many other species of bivalves with larval stages with similar sizes and shapes as larvae of M. mercenaria. In the past, when M. mercenaria was the dominant bivalve in this system, errors caused by misidentification may have been less significant. The apparent high abundance of other species coupled with the relatively low abundance of larvae of M. mercenaria presently makes estimates of hard clam larval abundance near impossible by using morphological criteria alone.
Although we found a similar relationship between larval shell length and width as that found by Loosanoff et al. (1966) (Fig. 2), the scatter around the regression line is indicative of the varied shapes of larvae of M. mercenaria. Because the hard clam larvae do not seem to strictly follow the isometric shape rule described by Loosanoff et al. (1966), it will be hard to distinguish them from other larval stages of bivalves.
Although morphological identification of hard clam larvae was found to be unreliable, this may not be the case for other species. Further studies are needed to test the accuracy of morphological identification of the larvae of other bivalves. The accurate and efficient identification of bivalve larvae is needed for accurate estimates of population production in the field, for monitoring sustainable fisheries, and for detection of populations at risk caused low reproduction rates. Accurate tools are also necessary for determining the success of conservation and restoration efforts. For M. mercenaria, molecular methods must be used for accurate estimates of larval densities and production in field populations.
This research was conducted in the Functional Ecology Research and Training Lab and the Molecular Evolution of Adaptation and Diversity Lab in the Ecology and Evolution department at Stony Brook University. Funding was provided by The Nature Conservancy, NOAA Community Based Restoration Program. For DKP, this paper was based on work supported by the National Science Foundation, while working at the Foundation. Any opinion, finding, and conclusions or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the National Science Foundation.
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LAURIE L. PERINO, (1) DIANNA K. PADILLA (2) * AND MICHAEL H. DOALL (2)
(1) Marine Science Research Center, Stony Brook University, Stony Brook, New York, 11794-5000; (2) Department of Ecology and Evolution, Stony Brook University, Stony Brook, New York, 11794-5245
* Corresponding author. E-mail: email@example.com
TABLE 1. Estimates of larval densities from plankton samples collected from Coecles Harbor (PB) and Great South Bay (GSB). Estimates were based on morphological identifications and by the use of molecular identification. No. of Total M. mercenaria bivalve x [L.sup.-1] larvae by Date Site x [L.sup.-1] morphology 6/30/04 PB 11.58 7.27 7/15/04 PB 41.67 16.83 7/30/04 PB 126.00 21.55 8/4/04 PB 15.28 3.56 7/14/04 GSB 5.11 0.00 7/14/04 GSB 8.50 0.20 7/27/04 GSB 9.33 2.89 8/3/04 GSB 4.56 0.50 8/10/04 GSB 2.00 0.00 8/17/04 GSB 2.44 0.88 No. of M. mercenaria x [L.sup.-1] N by for Date PCR PCR 6/30/04 0.00 39 7/15/04 0.00 40 7/30/04 6.17 41 8/4/04 0.37 83 7/14/04 0.11 29 7/14/04 0.29 46 7/27/04 0.29 32 8/3/04 0.00 38 8/10/04 0.00 34 8/17/04 0.08 31 TABLE 2. Error rates of morphological identification of Mercenaria mercenaria. Individual larvae were identified on the basis of morphology as either being M. mercenaria (+) or not M. mercenaria (-). Those same larvae were then tested for their molecular identification and were either found to be M. mercenaria (+) or not M. mercenaria (-). Molecular ID Morphological ID + 0 71 - 2 138
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