Within Carpilius Convexus
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Investigating Possible Evolutionarily Significant Units (ESUs) Within Carpilius convexus (Arthropoda: Malacostraca: Decapoda: Carpiliidae) An honors thesis submitted for graduating with honors from the University of Florida Biology Department Jennifer Mariko Thompson [email protected] UFID: Redacted Advisors: Nathaniel Evans, Gustav Paulay Division of Invertebrate Zoology Florida Museum of Natural History University of Florida Submitted: 27 March 2014 Thompson 2 Abstract Carpilius is a genus of large and brightly colored reef associated crabs comprised of the three species C. corallinus, C. maculatus, and C. convexus. The systematics of Carpilius crabs has not been thoroughly investigated and it is clear that significant taxonomic work is still needed. Previous efforts by Wetzer et al. (2003) suggested that the species C. convexus may actually consist of multiple genetically distinct lineages. To investigate this further, we generated and analyzed sequence data from the mitochondrial CO1 gene for 42 C. convexus specimens from localities throughout the native range of the species in the Indo-West Pacific. Mitochondrial 16S ribosomal sequence data was also generated to increase phylogenetic resolution and incorporate existing, published sequence data on C. convexus. Phylogenetic analyses of all this data suggest that the species C. convexus consists of at least two genetically distinct lineages that appear to exist in sympatry. One lineage appears to have significant phylogeographic structure and is comprised of five mostly allopatrically distributed evolutionary significant units (ESUs). The other lineage appears to be rare (or rarely collected) and is widely distributed from at least Indonesia to the Society Islands. Although all available specimens were examined, no significant morphological differences could be found in the first lineage. For the second lineage no adult specimens were available for morphological work. Thompson 3 Introduction Carpilius is a reef associated crab genus whose members are large, brightly colored, and appear to be most active at night (Wetzer et al. 2003). The genus remains poorly studied, but recent work by Wetzer et al. (2003), has confirmed that Carpilius is comprised of at least three genetically and morphologically distinct lineages, consistent with the three currently described species. These three species are C. corallinus, C. maculatus, and C. convexus. C. corallinus is bright reddish-orange with white speckling and ranges from the tropical western Atlantic Ocean (See Figure 1A). The other two species, C. maculatus and C. convexus, share a native range that spans the entire tropical Indo-West Pacific region. These two species can be easily distinguished from one another by their appearance. C. maculatus, sometimes called the 7-11 crab, is typically lightly colored with eleven large dark red spots with only seven of the spots observable when viewing head on (See Figure 1B). An adult C. maculatus is about 60mm to 120mm wide which is typically larger than an adult C. convexus which is about 50mm to 80mm wide. C. convexus is bright reddish-orange with the coloration pattern being either splotched or solid (See Figure 1C). Results presented by Wetzer et al. (2003) suggest that systematic and taxonomic work is especially needed for C. convexus. The authors found that C. convexus had significant intraspecific genetic structure, raising the possibility of multiple species. Here we follow up on the work of Wetzer et al. (2003) and focus on C. convexus. We investigate whether or not this species is actually comprised of multiple, distinct evolutionary significant units (ESUs) worth consideration as distinct species. To accomplish this we have examined C. convexus specimens from various locations throughout its native range to determine whether there are genetic differences among locations and whether there are distinct morphological differences between them. Genetic information offers the opportunity to test, in a phylogenetic context, whether Thompson 4 geographic and morphological groupings correspond to genetic groupings. Morphologically distinct groups that maintain genetic differentiation in sympatry meet the definition of biological species and we consider this in our evaluation. Crabs from the genus Carpilius are an important part of many coral reef ecosystems and are of economic value in some regions. With limited funding to support conservation, understanding species limits, distributions, and evolutionary history is important in setting conservation priorities. Our results provide important data for taxonomists to determine species delimitation in C. convexus. Methods Specimen Collection All 56 specimens for this project were sampled from the Florida Museum of Natural History collections where they are preserved in 75% ethanol. Locality information can be found in Table 1 and online <http://www.flmnh.ufl.edu/scripts/dbs/malacol_pub.asp>. DNA Extraction Two different methods of DNA extraction and purification were used. Initially, chelex extractions were used following the manufacturer’s protocol. Any extractions that did not work were repeated with new tissue from the same specimen using the Omega Biotek E.Z.N.A. Mollusc DNA Kit following the manufacturer’s protocol as well. Selection of DNA Markers An ~650 base pair portion of the mitochondrial gene CO1 was chosen for amplification and sequencing. This marker has been widely used to detect inter- and intraspecific genetic Thompson 5 variation due to its fast rate of sequence evolution (Hebert et al. 2003). Additionally, an ~600 base pair portion of the mitochondrial ribosomal (rDNA) gene 16S was amplified and sequenced for representatives of each distinct CO1 lineage of C. convexus found. This marker provided greater phylogenetic resolution between putative ESUs of C. convexus and enabled us to combine our data with published C. convexus sequence data including that of Wetzer et al. (2003). DNA Amplification To amplify the CO1 gene, we used either the dgHCO/dgLCO (Meyer 2003) or the jgHCO/jgLCO (Geller 2013) primer set. For both of these primer sets, a temperature of 42ºC was used for amplification. To amplify the 16S gene, we used either the 16H11/16L2 (Schubart 2009) primer set with an annealing temperature stepping between 48ºC, 42ºC, and 44ºC, or the Crust16F1/Crust16R2 (unpublished) primer set with an annealing temperature stepping between 55ºC, 52ºC, and 49ºC. Sequencing After each PCR reaction, the PCR products were visualized through gel electrophoresis. Successful amplifications were sent out to the University of Florida Interdisciplinary Center for Biotechnology Research for sequencing. Sequence data, Alignments and Phylogenetic Analyses Trace files of generated sequence data were assembled and edited into contigs of bidirectional reads in the software Geneious. Consensus sequences were generated for each contig and used in all further analyses. Sequences were aligned using MAFFT and phylogenetic Thompson 6 trees were constructed using a maximum likelihood approach as implemented in RAxML. This included a bootstrap analysis with 100 resampling replicate searches. Both CO1 and 16S rDNA data were analyzed separately and in a combined dataset. Combined analyses included 16S and 12S rDNA data from Wetzer et al. (2003). All sequences, including previously published data, analyzed in this study appear in Table 1. Results Of the 56 specimens which had their DNA extracted, only 42 resulted in successful PCR amplifications. PCR products for these specimens were sent out for sequencing. Separate phylogenetic analyses of CO1 and 16S rDNA data produced consistent topologies with one another and with our combined analyses (topologies not shown). Our combined analysis of CO1, 16S rDNA, and 12S rDNA sequence data (see Figure 2) produced a topology that suggests there are two major genetically distinct lineages of C. convexus that appear to exist in sympatry. One lineage (Lineage 1, Figure 2) appears to have significant phylogeographic structure represented by five mostly allopatrically distributed ESUs (see Figure 3). Phylogenetic divergence between these ESUs are all well supported but suggest a complex history where geographical proximity does not predict sister relationships of ESUs. Although each specimen was examined, no significant morphological differences were found between ESUs. Only a single ESU was recovered in the other C. convexus lineage (Lineage 2, Figure 2), which appears to be rare (or rarely sampled) and is widely distributed from at least Indonesia to the Society Islands (see Figure 3). For this lineage, only larval specimens were available for sequencing. As a result, no adult specimens were available for morphological examination. However, previously published CO1 data from an adult C. convexus specimen fell within Lineage 2. Thus, future work should Thompson 7 include examination of this specimen which was collected in Indonesia and accessioned at Singapore’s Raffles Museum (ZRC 2008.0174). Discussion Our analyses reveal the existence of at least two genetically distinct lineages of C. convexus, strongly suggesting that this species is actually comprised of at least two species living in sympatry. However, both lineages will require further investigation to clarify their taxonomic status. For Lineage 1, further morphological work is needed to determine if it is comprised of multiple mostly allopatrically distributed species. For example, the gonopods from each ESU need to be compared and examined for differences.