Cryptic Species and Synonyms: A Reclassification of the Tropical Spurilla Genus
Part of the Young Naturalist Awards Curriculum Collection.
INTRODUCTION
Shafts of sunlight pierce through the lush mangrove foliage and glimmer off the rippling pools of water. In a vibrant yellow kayak discordant with the serene sanctuary, where fishes of quicksilver zip through the natural maze of gnarled roots and curious crustaceans peep their eyestalks through the loamy sand, I cautiously maneuver through a remarkable ecosystem teeming with life. I have journeyed far from my home in the valleys of California to the Gulf of Mexico in search of only one creature: a rosy sea slug of the genus Spurilla, with gaudily beautiful extensions known as cerata.
Spurilla neapolitana (Delle Chiaje 1841) is a widely recognized aeolid due to its circumtropical distribution and exceptional survival abilities. As of today, specimens have been described in Morocco, Senegal, the Cape Verde Islands, the Sargasso Sea, the Mediterranean, the French Atlantic coast, Hawaii, Florida, Texas, Jamaica, Brazil, Belize, Honduras, Costa Rica, Colombia, Venezuela, the Bahamas, Bermuda, Puerto Rico, Curacao, and Barbados (Dominguez et al. 2007, Marcus 1960). The species is known for its singular ability to harbor in its cerata both nematocysts and zooxanthellae from consumed Aiptasia sea anemones (Conklin and Mariscal 1977, Rudman 1982). The nematocysts are used as a defense mechanism, and the zooxanthellae supplement its diet by carrying out photosynthesis. S. neapolitana is also an extremely variable species, with a diverse palette of hues. Its background coloration ranges from rosy pink to creamy orange, with opaque white pigmentations that form a variety of unique patterns (Valdes et al. 2006). Due to the breadth of its distribution and coloration, the species possesses an inordinate number of synonyms.
One of the species closely related to Spurilla neapolitana is S. sargassicola. First described by Kroyer in 1861, it has since been assumed, with shallow morphological data, to be a synonym of S. neapolitana (Gosliner 1980). In the present day, it is confusingly documented as both a synonym and a separate species. S. sargassicola is found on Sargassum algae, from which it derives its name, in only the Bahamas and the Sargasso Sea. While this species is pelagic, S. neapolitana has thus far been described as solely benthic.
The intent of my project is twofold: (1) to conclude whether Spurilla sargassicola is a synonym of S. neapolitana, or warrants categorization as a different species; and (2) to determine whether any cryptic species of S. neapolitana exist. The H3 histone and 16S rRNA genes, never obtained for any species in the Aeolidiidae family, will serve as the basis for analysis. Dissection of diagnostically reliable features, including the radula, jaws and reproductive organs, will also be used to differentiate between the species.
MATERIALS AND METHODS
Morphological Examination
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Specimens, accessed through the Natural History Museum of Los Angeles County (LACM), were collected in the tropical eastern Pacific and western Atlantic oceans (Table 1 and Figure 2). My dissection of the specimens was facilitated by a dissecting microscope with a camera lucida (Figure 1). Starting at the anterior end of the slug, the tissue was divided with a pair of sharp forceps to gain entry to the interior. The buccal mass, the muscular pouch where the radula is situated, and the jaws surrounding the mass, were extracted and placed in 10% sodium hydroxide for two hours until some tissue is dissolved. The jaws were then carefully removed and mounted on a copper surface for the scanning electron microscope (SEM). The remaining buccal mass was returned to the NaOH until all the soft tissue was dissolved, isolating the radula. The radulae were then rinsed in distilled water and mounted on a copper surface for the SEM.
The male and female copulatory organs are located directly underneath the jaws and buccal mass. The organs were detached from the body wall and the digestive glands, and drawn with the camera lucida lens. The penis, prostate, ampulla, and female glands were compared, with special attention paid to the seminal receptacle.
All SEM analysis was conducted at the LACM facility. Mounted gizzard plates and radulae were sputter-coated with gold and palladium with an EMITECH K-550X sputter coater for four minutes to create a conductive surface. The coated samples were then observed under an S-3000N Hitachi SEM in a high vacuum chamber at 10 kilovolts. Photomicrographs were taken digitally and saved for further examination.
DNA Analysis
DNA was extracted from a finely cut 3mm piece of non-cerata tissue preserved in 70% ethyl alcohol. After incubation in 1ml of TE buffer at room temperature for 15 minutes, samples were centrifuged so that 975µl of supernatant could be removed and 175 µl of 10% Chelex added. The mixture was then incubated at 56°C for 20 minutes and 100°C for 8 minutes. After centrifuging, 20µl of the supernatant can be used for polymerase chain reaction (PCR).
For the PCR, I amplified portions of 16S rRNA genes using Taq polymerase, a thermostable enzyme, and the primers 16Sar and 16Sbr. I amplified the H3 histone gene using the primers H3AF and H3AR. The cycle consisted of two minutes initial denaturation at 94°C, 30 seconds denaturation at 94°C, 30 seconds annealing at 50°C, 60 seconds extension at 72°C, and 120 seconds of final extension at 72°C. The steps of denaturation, annealing, and extension were repeated 30 times for the 16S rRNA and 40 times for the H3 histone genes before final extension. To ascertain the presence of DNA in the sample after PCR, I used a 1.0% agarose gel electrophoresis test. Successful PCR samples were cleaned with Promega PCR Clean-Up System and diluted in preparation for sequencing.
After sequencing at the City of Hope Beckman Research Institute, I utilized the computer programs Geneious and Free Work Bench to analyze and compare the sequences. All the sequences will be submitted to GenBank.
RESULTS
External Morphology
The coloration of all the specimens was generally consistent: an ivory background with opaque white pigmentation covering the head, back and cerata. The only exception was Spurilla sargassicola, which possesses unique dark-brown patterns on its entire body (Figure 3A), and the Pacific S. neapolitana, which tends to have a more pink coloration (Figure 3C). The rhinophores of all the specimens were perfoliate, with many lamellae, and the thick cerata were curved at the apex and translucent, with visible pale-brown digestive glands. Oral tentacles of the same body coloration were elongate and tapering.
Reproductive Organs
The reproductive organs were the same overall shape for all the specimens, with a tapering ampulla that bifurcates into the bursa copulatrix and the large female glands. The vas deferens gradually enlarges to form a large looped prostate leading into the penis. The bursa copulatrix is simple and short for the Bahamas population of S. neapolitana and the Pacific Spurilla neapolitana, but folded and convoluted for S. sargassicola (Figure 4).
Jaws
The strong, ovoid jaws possessed a completely smooth masticatory border, with no variation between specimens (Figure 5).
Radula
The uniserate and broadly arched radula proved to be a major differentiating characteristic. The pectinate teeth consist of short, triangular central cusps flanked by elongated lateral denticles. I conducted the morphometric analyses with a nonparametric Mann-Whitney U-test, since the data were not normally distributed. The test showed that the population of Spurilla neapolitana at Stocking Island, Bahamas, has significantly (P=0.01) longer
and slimmer central cusps (ratio of cusp width to length), longer and slimmer lateral denticles (ratio of lateral width to length), and wider denticles than cusps (ratios of cusp width to lateral width) when compared to all other specimens (Figure 6D). All other Pacific (Figure 6C) and Atlantic (Figure 6B) S. neapolitana as well as S. sargassicola (Figure 6A) have consistent radular morphology.
H3 Sequences
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H3 sequences were obtained for specimens from across all groups: Spurilla sargassicola, the Atlantic S. neapolitana, and the Pacific S. neapolitana. Surprisingly, the group that was most divergent among the sequences was not S. sargassicola, but rather one population of S. neapolitana in Stocking Island, Bahamas. This population differs in five base pairs out of 328 (a 1.52% sequence divergence), while all the other sequences, including those of S. sargassicola, vary in one base pair or less. When maximum likelihood trees with bootstrap values of 100 are constructed from sequence data, it is clearly evident that the Stocking Island population of S. neapolitana forms an exclusive clade, and that S. sargassicola groups with all other S. neapolitana sequences (Figure 7).
16S Sequences
Although I had greater efficacy obtaining H3 gene sequences from the specimens than 16S sequences, the garnered 16S sequences, from which only Spurilla sargassicola is missing, confirm the H3 sequence findings. The Bahamas S. neapolitana population differs in 28 out of 440 base pairs, resulting in a 6.36% sequence divergence, while other S. neapolitana sequences have a maximum 2.50% sequence divergence. The maximum likelihood trees constructed with the 16S data also show that the Stocking Island population of S. neapolitana forms an exclusive clade (Figure 8).
DISCUSSION
The thorough analysis conducted in this research brought to light which characteristics are efficacious for differentiating species within the Spurilla genus. The majority of morphological variations are based in the radula, which had been noted as a variable characteristic in Spurilla neapolitana (Garcia-Gomez & Thompson 1989). However, with the range of specimens from two oceans showing that the radula retains the same morphology in all but one population, it is determined here to be an ideal feature for comparison. The bursa copulatrix of the reproductive system is also identified as a source of fluctuation. In the Atlantic, only the Bahamas population of S. neapolitana exhibits a simple bursa copulatrix, while S. sargassicola and other S. neapolitana have a folded structure. Interestingly, the Pacific specimens also possess the simple structure, suggesting perhaps the more convoluted bursa copulatrix evolved to aid copulation. While R.S. Bergh (1877) and I. Hamatami (2000) found that the masticatory border of S. neapolitana can have serration, all the jaws in this project had smooth borders consistent with the findings of Dominguez et al. (2007) and Garcia and Cervera (1985). Thus, jaws have been shown to not be useful in distinguishing between any of the populations.
The hypervariable 16S rRNA gene is ideal for phylogenetic studies because it provides specific signature sequences for species. However, as was found in previous studies and again in this research, it is very difficult to obtain 16S sequences. Thus, in addition to 16S rRNA, H3 genes were also sequenced. Although the H3 histone gene is highly conserved due to its integral role in the structure of chromatin, its sequences were still useful in determining the extent of differentiation between specimens. The differences in the base pairs were all determined to be silent mutations when translated to the amino acid sequences, but the selection of particular codons, even when there is no change to the amino acid sequence, affects translational stability. Since H3 is a nuclear gene—as opposed to 16S, which is mitochondrial—it serves not only as a verification of genetic data but also checks for occurrences such as hybridization, which can show up in the 16S gene. In this case, since the two sets of genetic data corroborated each other, no hybridization has occurred.
From the gathered morphological and molecular data, it is indisputable that Spurilla sargassicola is a synonym of S. neapolitana and should not retain its separate name. Although there are small differences in the sea slug's external morphology and habitat preference, H3 sequences reveal that it is nearly identical to its S. neapolitana counterparts, even in the Pacific. All aspects of its morphology, including the radula, jaw, and reproductive organs, are also identical to those of S. neapolitana. Thus, S. neapolitana is now identified as both a benthic and a pelagic species.
It is also evident that the Stocking Island population of Spurilla neapolitana is undergoing a speciation event, as it is more divergent from its fellow Atlantic specimens than even the Pacific specimens are (Figures 7 and 8). Further research requires the obtainment of additional specimens. To determine whether the extent of the divergence is sufficient for this population to be considered a separate species, sequences from an outgroup, preferably within the same family and ideally within the same genus, are in the process of being procured. Since the type locality of S. neapolitana is from Naples, Italy, European Spurilla neapolitana specimens are also necessary to determine whether only the divergent Bahamas population or all the other tropical western Atlantic and eastern Pacific specimens will be given a new name. It is also possible that both are divergent enough from the true S. neapolitana and each other that they are both new species. Since the species' distribution is not restricted to the area studied in this project, there may be more cryptic species or cases of synonyms that will be found when the sample area is expanded to include specimens from S. neapolitana's entire range.
Interestingly, while Spurilla sargassicola is now determined to be S. neapolitana, the originally described S. neapolitana from the exact same locale is now identified as a highly likely cryptic species. Since the major source of morphological difference was found in the radula, a possible reason for such a sympatric speciation event can be due to the fact they are exposed to different sources of food. Variable feeding habits are already known to be a cause of the wide range of color that S. neapolitana possesses (Haefelfinger 1969), thus it is likely that they can also lead to speciation.
A crucial part of this research is gaining insight into the understanding and conservation of biodiversity. With the escalating loss of marine species comes a loss of stability and productivity in entire ecosystems. However, it will be impossible to protect these species unless a lucid picture of the distribution, genetic differences, and uniqueness of the populations alive today can be provided.
ACKNOWLEDGEMENTS
First and foremost, an expression of gratitude to my mentor, Dr. Ángel Valdés of California State Polytechnic University at Pomona, for his invaluable research guidance, and to graduate student Elysse Gatdula for her aid in DNA procedures. Thanks are also due to LACM for permitting access to its specimen collections and the SEM lab. This study would not have been possible without their generous assistance.
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Valdés, Á., et al. Caribbean Sea Slugs: A Field Guide to the Opisthobranch Mollusks from the Tropical Northwestern Atlantic. Global Interprint Inc.: Hong Kong, 2006.
Figure 3 photos by Anne Dupont and Angel Valdes. Used with permission.