ArticlesMorphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaThomas Sauvage Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, USACorrespondencetomsauv@gmail.com, Michael J. Wynne University of Michigan Herbarium, 3600 Varsity Dr., Ann Arbor, MI 48108, USA, Valerie J. Paul Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Fort Pierce, FL 34949, USA Suzanne Fredericq Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, USAMorphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaIntroductionOngoing biodiversity assessment of the Caulerpaceae from the western Atlantic triggered an investigation of the enigmatic taxon known as C. floridana W.R. Taylor (1960) from the Dry Tortugas (Monroe County, Florida). In the summers of 1924, 1925 and 1926, Taylor (1928) dredged from depths of 18–109 m a set of specimens resembling C. ashmeadii Harvey (1858), an endemic species commonly found in the region, which at the time he considered as representing a ‘perfect and elegant form’ of that species (Taylor, 1960). Later, following the opinion of M.A. Howe, who had borrowed Taylor’s collections and indicated his intention to publish the specimens as a new species (C. taylorii M. Howe) but did not do so prior to his death in 1936, Taylor described C. floridana based on contrasting features of the branchlets (i.e. pinnules). In his treatment of Caulerpa, Taylor (1960) reported the pinnules of C. floridana as acuminate, compressed and basally constricted, and those of C. ashmeadii as obtuse and terete throughout (i.e. lacking an apiculus), characters that may be easily overlooked due to the convergent habit of both species.Since the time of this description, there have been no new records of C. floridana from Florida (Dawes Mathieson, 2008) or elsewhere in the Gulf of Mexico (Fredericq et al., 2009; Wynne, 2011). Earle (1972) omitted this species from her list of benthic marine algae found in the Gulf, and Dawes Mathieson (2008) referred to it as ‘rare’ in Florida. However, new records were made in the Caribbean Sea by Prado Díaz Suárez (1997) from the southeastern platform of Cuba, and by Joly et al. (1976) from dredged depths of 37 m and 49 m at sites off the coast of the States of Ceará and Bahia, Brazil, although the morphological circumscription of these distantly located records with the Dry Tortugas specimens remains to be ascertained. By comparison, specimens identified as C. ashmeadii have been reported from the Gulf of Mexico, in Florida (Taylor, 1960) and the Mexican States of Yucatan and Quintana Roo (Taylor, 1941; Huerta et al., 1987; Mendoza González Mateo Cid, 1992; Ortega et al., 2001), within the Antillean island arc (i.e. Greater and Lesser Antilles) in Cuba (Díaz Piferrer, 1964), the Virgin Islands (Børgesen, 1913), Puerto Rico (Ballantine, 1997), and Nevis (Taylor, 1969), and in the southwestern Atlantic in the Venezuelan state of Mirando (Rios, 1972) and the Brazilian state of Pernambuco (Taylor, 1930). Interestingly, neither C. ashmeadii nor C. floridana has been recorded from the Caribbean coast of Central America (e.g. no record in Wysor Kooistra, 2003).In spite of its enigmatic identity, Caulerpa floridana is currently banned from the State of California (importation, sell and trade, DFG Code 2300) since the C. taxifolia (H. West) C. Agardh invasion of the Agua Hedionda Lagoon, Carlsbad, California (Jousson et al., 2000), and its subsequent eradication (SCCAT, 2006) (note that according to Art. 46 of the International Code of Nomenclature (McNeill et al., 2012), C. taxifolia and C. cupressoides have to be ascribed to H. West rather than M. Vahl; cf. Nielsen Price 2001 and Wynne 2011). Eight additional Caulerpa species are banned from California, partly for their proven invasive potential elsewhere, i.e. the non-pinnate species C. racemosa (Forsskål) J. Agardh sensu lato including the variety cylindracea spreading in the Mediterranean Sea (e.g. Klein Verlaque, 2008) (now C. cylindracea Sonder, fide Belton et al., 2014) and C. verticillata J. Agardh in Florida (Lapointe et al., 2005) and the Gulf of California, Mexico (Pérez-Estrada et al., 2013), and pinnate, feather-like species that may be easily confused with C. taxifolia: C. ashmeadii, C. cupressoides (H. West) C. Agardh, C. mexicana Sonder ex Kützing, C. scalpelliformis (Turner) C. Agardh and C. sertularioides (S.G. Gmelin) M. Howe. In an informative pamphlet prepared by Merkel Associates, Inc., illustrating the above taxa and entitled ‘A guide to the nine species of Caulerpa banned in California’ (available at www.cnpssd.org/CaulerpaBrochure.pdf), the photographed field specimen of C. floridana by Vincent B. Hargreaves does not seem correctly identified in light of Taylor’s description (1960) and our preliminary observations of the type material made at the University of Michigan Herbarium (MICH). In this context, molecular and morphological data are urgently needed to confirm the identity and separation of C. floridana from C. ashmeadii, as it applies to conservation issues such as ban effectiveness (Diaz et al., 2012) and invasive species management in the State of California (State of California Resources Agency, 2008). Moreover, C. floridana may hold subjective intrinsic value (Sandler, 2012) as an endemic species of the western Atlantic, and thus deserves further clarification as a potential member of this unique regional biotic diversity (Soulé, 1985).The submarine regions around the Dry Tortugas have become less accessible for exploration since their increased protection as a national park (26 October 1992) bordering two Ecological Reserves of the Florida Keys Marine National Sanctuary (since 1 July 2001). Thus, current logistics led to the study of historical material. DNA sequencing of herbarium algal specimens from the 19th and 20th century for the Rhodophyta (e.g. Hughey et al., 2001; Gabrielson, 2008a, b; Hughey Gabrielson, 2012) or the Chlorophyta (Provan et al., 2008; Carlile et al., 2011) have typically relied on the amplification of short DNA fragments (i.e. amplicons) of 100–200 bp because of the degradation of DNA into short strands through time (e.g. Staats et al., 2011, 2013). These studies have sought to provide molecular identification/confirmation of historical specimens with single (unique) amplicons for DNA markers such as rbcL (chloroplast), COI (mitochondrial) and 18S, ITS1 and ITS2 (nuclear). Among these studies, Provan et al. (2008) generated multiple, overlapping short amplicons (rather than a single amplicon per DNA marker) to reconstruct longer sequences of 360 bp of the chloroplast gene rpl16-rps3 from numerous herbarium specimens of Codium fragile (Suringar) Hariot and subspecies in order to gain further phylogenetic signals to sort out cryptic entities and invasive haplotypes in this complex. A similar PCR strategy (i.e. overlapping short amplicons) had been adopted earlier by e.g. Krings et al. (1997) to reconstruct a ~400 bp from a Neanderthal’s humerus ( 30 000 years old), and later by e.g. Alonso et al. (2003) in a multiplex fashion to obtain 400 bp from Homo sapiens bone remains ( 4000 years old), and by Heupink et al. (2011) to recover a long stretch of COI ( 1500 bp) from subfossil remains ( 14 000 years old), also in a multiplex way.In the present study, we sequenced the chloroplast gene tufA from herbarium specimens of C. floridana dating from 1924 (WRT329, WRT345 and WRT349), which W.R. Taylor had collected along with the holotype of the species (WRT361). We reveal the genetic identity and phylogeny of C. floridana based on the reconstruction of 931 base pairs of tufA after the assembly of short overlapping amplicons generated from the historical, degraded DNA. In addition, we review the morphological circumscription of C. floridana specimens recorded in the southwestern Atlantic from Brazil (Joly et al., 1976).Materials and methodsHistorical DNA from herbarium specimens is typically degraded to short strands of 100 to 400 bp (e.g. Staats et al., 2011, 2013) preventing the direct amplification of ~820–920 base pairs of tufA with routine primers (e.g. Famà et al., 2002). Thus, we designed a series of Caulerpa-specific primers to amplify overlapping short fragments in order to reconstruct the span of the gene tufA (Fig. 1). Primer design performed with Primo Pro 3.4 (Chang Bioscience) relied on a previously compiled sequence alignment of the genus Caulerpa to examine conserved regions and/or character variation at potential priming sites. From this analysis, 12 primers (six pairs) were designed to amplify six overlapping fragments of length 226 to 316 bp which, after assembly, resulted in reconstructed sequences of a maximum length of 931 bp for phylogenetic analyses. The newly designed primers were tu157F-tu391R (amplicon of 234 bp), tu283F-tu599R (316 bp), tu459F-tu686R (226 bp), tu585F-tu818R (233 bp), tu755F-tu1017R (262 bp) and tu853F-tu1118R (265 bp) (Table 1). Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 1. Position of newly designed primers and generated overlapping amplicons for WRT329 (top), WRT345 (middle) and WRT349 (bottom) used to reconstruct up to 931 bp of tufA for C. floridana (Red: Thymine, Blue: Cytosine, Green: Adenine, Purple: Guanine). Note that amplicon tu157F-tu391R could not be amplified for WRT349 and is thus missing. Primer names are based on their position on the Ostreococcus tauri Courtes Crétiennot complete tufA sequence (CR954199). Open boxes correspond to the multiple sequence alignments detailed in Figs 13–14.Display full sizeFig. 1. Position of newly designed primers and generated overlapping amplicons for WRT329 (top), WRT345 (middle) and WRT349 (bottom) used to reconstruct up to 931 bp of tufA for C. floridana (Red: Thymine, Blue: Cytosine, Green: Adenine, Purple: Guanine). Note that amplicon tu157F-tu391R could not be amplified for WRT349 and is thus missing. Primer names are based on their position on the Ostreococcus tauri Courtes Crétiennot complete tufA sequence (CR954199). Open boxes correspond to the multiple sequence alignments detailed in Figs 13–14. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Table 1. Newly designed primers for the present study with position based on the Ostreococcus tauri Courtes Crétiennot complete tufA sequence (CR954199). GC: Guanine-Cytosine content; Tm: melting temperature; bp: primer length in base pairs.CSVDisplay TableSeveral herbarium specimens of C. floridana housed at the University of Michigan Herbarium (MICH) (Table 2) were photographed (Figs 2–6), including the holotype WRT361 (Fig. 2) and its isotype maintained at the New York Botanical Garden (NY) (image downloaded from the C.V. Starr Virtual Herbarium at NY, Fig. 3), and samples of 6–8 pinnules per specimen were taken from the sheets of WRT329, WRT345 and WRT349 (Figs 4–6). Excised branchlets from each of the specimens were ground separately with new (sterile) pestles in 1.5 ml tubes, and their DNA extracted according to the modified protocol of Dellaporta et al. (1983) described in Hughey et al. (2001) with fresh (never used prior) buffers prepared in previously chlorinated containers. During extraction steps in the Seaweeds Lab at UL Lafayette, all liquids (e.g. buffers) were handled with UV-sterilized pipettes and barrier tips, and powder-free nitrile gloves changed between sample manipulations. Prepared DNA extracts were then stored at −20°C in separate zip bags until processing and between uses to avoid cross-contamination. All subsequent PCR steps were conducted in a separate Microbiology laboratory (Dr Chistoserdov, UL Lafayette), where manipulations were performed under a laminar flow hood equipped with a germicidal lamp for bench sterilization, and a UV-cross linker box (254 nm output, UVC-515, Ultra-Lum Inc., Claremont, California, USA) for DNA decontamination (i.e. cross-linking of any contaminant DNA) of pipette tips and pipettors, as well as tubing and PCR reagents (MangoTaqTM kit, Bioline USA Inc., Taunton, Massachusetts, USA), according to the protocol and recommendations of Champlot et al. (2010). Briefly, a first PCR mix containing 5X Mango Buffer, MgCl2 and molecular grade H2O was prepared following the manufacturers’ protocol, and UV-sterilized for 10 minutes in 0.2 ml polypropylene PCR tubes with caps placed at 10 cm from the UV-source. Then, a second mix consisting of the MangoTaq, ultrapure dNTPs (Bioline USA Inc.) and PCR primers (previously diluted in UV-sterilized H2O and tubing) was assembled and distributed among PCR tubes containing the first mix. Finally, 1 µl of the corresponding DNA extracts diluted to 1:100 (in UV-sterilized H2O and tubing) was pipetted into their corresponding PCR tubes. Each PCR reaction (i.e. a well with DNA template) was directly preceded and followed by a negative PCR control (no DNA template) reaction to assess for reagents and/or manipulation contaminations. Powder-free nitrile gloves were changed regularly throughout the different PCR steps. When contaminated, PCRs were repeated and reagents replaced. PCR cycling consisted of a low temperature-long annealing cycle starting at 95°C for 3 min, followed by 40 cycles at 94°C for 1 min, 42°C for 1 min and 72°C for 1 min, and with a final extension of 5 min at 72°C. PCR products were visualized on a 0.1% TBE agarose gel with extended running time until separation from primer dimers. Products of correct sizes were purified using ExoSAP-IT (Affymetrix, Inc., Cleveland, Ohio, USA) and cycle-sequenced in both directions with BDX64 enhancing buffer (Molecular cloning Lab (McLab), San Francisco, California, USA) and BigDye® 3.1 (Life Technologies Corporation, Grand Island, New York, USA) following McLab’s 64-fold dilution protocol (0.875 µl BDX64, 0.125 µl BigDye® Terminator 3.1, 1.5 µl BigDye® 5X Sequencing Buffer, 1.5 µl of 3.2 pmol primer, 1.5 µl DNA template and 5.0 µl H2O) and recommended cycling conditions (96°C for 3 min, 30 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 2 min). Cycle sequenced products were purified by alcohol precipitation, vacuum dried, resuspended in 20 µl Hi-DiTM Formamide (Life Technologies Corporation) and loaded on the UL Lafayette ABI Prism® 3130XL Genetic Analyzer (Life Technologies Corporation) for sequencing. Individual chromatograms were assembled into contigs, cropped from incorporated primers and further edited using Sequencher v.5.1 (Gene Codes, Ann Arbor, Michigan, USA). Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Table 2. Herbarium specimens of C. floridana sensu stricto present in MICH and examined in the present study. All originally collected from the Dry Tortugas, White Shoal, and off Southwest Channel.CSVDisplay Table Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Figs 2–3. Holotype and isotype of Caulerpa floridana W.R. Taylor. Fig. 2. Holotype of WRT361 maintained at MICH. Note early identification as C. ashmeadii Harvey. Fig. 3. Isotype of WRT361 maintained at NY. Note early identification as C. ashmeadii Harvey, later corrected as C. taylori M.A. Howe. Scale bars: 1 cm (Figs 2–3).Display full sizeFigs 2–3. Holotype and isotype of Caulerpa floridana W.R. Taylor. Fig. 2. Holotype of WRT361 maintained at MICH. Note early identification as C. ashmeadii Harvey. Fig. 3. Isotype of WRT361 maintained at NY. Note early identification as C. ashmeadii Harvey, later corrected as C. taylori M.A. Howe. Scale bars: 1 cm (Figs 2–3). Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Figs 4–6. Historical herbarium specimens of Caulerpa floridana W.R. Taylor sampled for molecular assessment (note early identification as C. ashmeadii). Fig. 4. Specimen WRT349. Fig. 5. Specimen WRT329. Fig. 6. Specimen WRT345. All are maintained at MICH. Scale bars: 1 cm (Figs 4–6).Display full sizeFigs 4–6. Historical herbarium specimens of Caulerpa floridana W.R. Taylor sampled for molecular assessment (note early identification as C. ashmeadii). Fig. 4. Specimen WRT349. Fig. 5. Specimen WRT329. Fig. 6. Specimen WRT345. All are maintained at MICH. Scale bars: 1 cm (Figs 4–6).The three newly reconstructed tufA sequences (WRT329, WRT345 and WRT349 corresponding to GenBank accession KF921071, KF921072 and KF921073, respectively) were added to the sorted alignment of Sauvage et al. (2013) with additional outgroup taxa (Dasycladales FJ535854, FJ535855, FJ535858; Dichotomosiphonaceae FJ432651, FJ432652; early branching Caulerpa flexilis AJ417970 and C. verticillata AJ417967; and Caulerpa with pyrenoid-containing chloroplasts, i.e. C. cactoides AJ417969 and C. bartoniae FJ810426). A newly generated sequence of C. ashmeadii extracted and amplified with routine tufA primers at SMS, Fort Pierce, was also included (see Fig. 7 for specimen illustration; TS1824 accessioned KF977086 from Long Key, N24°50′19.01′′, W80°47′46.00′′, vicinity of type locality), as well as two published miscellaneous sequences from the Mediterranean Sea, FM956042 C. prolifera (Forsskål) J.V. Lamouroux (courtesy Dr Stefano Draisma) and JX185603 C. taxifolia var. distichophylla (Sonder) Verlaque, Huisman Procaccini (Jongma et al., 2013). Among accessions for C. ashmeadii found on GenBank (Stam et al. 2006), redundant sequences were excluded as well as those of lower quality, as indicated by the presence of numerous ambiguous bases close to priming sites, pointing at chromatogram assembly issues (e.g. DQ652369). The phylogenetic analysis was run in RAxML v7.2.8 (Stamatakis, 2006) with a GTR+I+G model of evolution applied per codon position as indicated by PartitionFinder (Lanfear et al., 2012). RAxML topological searches were executed with the rapid hill-climbing algorithm and 200 restarts each from a random starting tree. Branch support was assessed with non-parametric bootstrapping of 1000 replicates, and the resulting bootstrap values plotted on the best tree (i.e. the topology with lowest likelihood value) (see Fig. 8). For figure clarity, outgroup taxa were pruned from the tree and several clades were collapsed in FigTree (Andrew Rambault, tree.bio.ed.ac.uk). Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 7. Herbarium sheet of Caulerpa ashmeadii Harvey (TS1824) newly collected from the Florida Keys (Long Key) for DNA sequencing and morphological illustration. Scale bar: 1 cm.Display full sizeFig. 7. Herbarium sheet of Caulerpa ashmeadii Harvey (TS1824) newly collected from the Florida Keys (Long Key) for DNA sequencing and morphological illustration. Scale bar: 1 cm. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 8. Phylogenetic position of Caulerpa floridana W.R. Taylor and closely related species based on reconstructed tufA sequences for the three historical herbarium specimens studied, viz. WRT329 (931 bp), WRT345 (927 bp) and WRT349 (771 bp). The topology and bootstrap support values (numbers above branches) were obtained with RAxML. Labels in grey shade correspond to newly generated sequences. Lineages A–F represent entities of the C. racemosa-peltata complex sensu Sauvage et al. (2013). Asterisks indicate alien strains of C. taxifolia.Display full sizeFig. 8. Phylogenetic position of Caulerpa floridana W.R. Taylor and closely related species based on reconstructed tufA sequences for the three historical herbarium specimens studied, viz. WRT329 (931 bp), WRT345 (927 bp) and WRT349 (771 bp). The topology and bootstrap support values (numbers above branches) were obtained with RAxML. Labels in grey shade correspond to newly generated sequences. Lineages A–F represent entities of the C. racemosa-peltata complex sensu Sauvage et al. (2013). Asterisks indicate alien strains of C. taxifolia.Finally, for morphological comparison, high-resolution scans of C. floridana specimens from Brazil (Joly et al., 1976) were provided by the Universidade de São Paulo herbarium (SPF) (courtesy of Viviane Jono) (see Fig. 9 for a variable specimen with two thalli commonly recorded as SPF04257; SPF04255 and SPF04256 not shown, and Table 3 for collection information). Unfortunately, the record of C. floridana from Cuba could not be examined (Prado Díaz Suárez, 1997), as it was not maintained in a permanent collection (Dr A. M. Suárez, Univ. de La Habana, personal communication). An additional specimen from Brazil collected off Pernambuco State by the Albatross Expedition of 1887 (Taylor, 1930) was encountered in the MICH collections during the present project and photographed (Fig. 10, Table 3). Although published by Taylor in 1930 as C. ashmeadii, it had been examined by M.A. Howe in 1929, and later annotated by W.R. Taylor as ‘C. taylori Howe inedit’ (i.e. C. floridana) for the occasional presence of apiculi (e.g. see arrow, Fig. 10). The recently collected specimen of C. ashmeadii from Long Key was also detailed to contrast its pinnules with those of C. floridana (Fig. 11). Finally, the unknown Caulerpa sp. previously collected from the Florida Middle Grounds (FMG), sequenced by Famà et al. (2002) (GenBank accession AJ417962) and maintained at the University of Louisiana at Lafayette herbarium (LAF) (accession 12.viii.00-1-52) was also photographed (Fig. 12) for documentation considering its sister relationship to the taxa investigated. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 9. Herbarium sheets of specimen SPF04257 reported as Caulerpa floridana W.R. Taylor by Joly et al. (1976) from Ceará State, Brazil. Note pencil markings ‘floridana ?’ and ‘ashmeadii ?’ on the left and right thalli, respectively. Specimen maintained at SPF. Scale bar: 1 cm.Display full sizeFig. 9. Herbarium sheets of specimen SPF04257 reported as Caulerpa floridana W.R. Taylor by Joly et al. (1976) from Ceará State, Brazil. Note pencil markings ‘floridana ?’ and ‘ashmeadii ?’ on the left and right thalli, respectively. Specimen maintained at SPF. Scale bar: 1 cm. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Table 3. Southwestern Atlantic specimens from Brazil reported as C. floridana or C. ashmeadii by Joly et al. (1976) and Taylor (1930).CSVDisplay Table Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 10. Herbarium sheets of specimen T13894 reported as Caulerpa ashmeadii Harvey by Taylor (1930) from Pernambuco State, Brazil. See annotation made by Taylor regarding ‘traces of apiculi’ and pointed arrow on right assimilator. Specimen maintained at MICH Scale bar: 1 cm.Display full sizeFig. 10. Herbarium sheets of specimen T13894 reported as Caulerpa ashmeadii Harvey by Taylor (1930) from Pernambuco State, Brazil. See annotation made by Taylor regarding ‘traces of apiculi’ and pointed arrow on right assimilator. Specimen maintained at MICH Scale bar: 1 cm. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 11. Assimilator and pinnules comparison for C. floridana W.R. Taylor (WRT329, top) and C. ashmeadii Harvey (TS1824, bottom). Abbreviations: ap: apiculi; co: constrictions. Scale bar: 0.5 cm.Display full sizeFig. 11. Assimilator and pinnules comparison for C. floridana W.R. Taylor (WRT329, top) and C. ashmeadii Harvey (TS1824, bottom). Abbreviations: ap: apiculi; co: constrictions. Scale bar: 0.5 cm. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Fig. 12. Herbarium sheet of the unknown Caulerpa sp. (12.viii.00-1-52, AJ417962) originating from the Florida Middle Grounds previously sequenced by Famà et al. (2003). Specimen maintained at LAF. Scale bar: 1 cm.Display full sizeFig. 12. Herbarium sheet of the unknown Caulerpa sp. (12.viii.00-1-52, AJ417962) originating from the Florida Middle Grounds previously sequenced by Famà et al. (2003). Specimen maintained at LAF. Scale bar: 1 cm.ResultsPCR amplifications targeting short overlapping fragments allowed the successful reconstruction of the gene tufA from historical C. floridana specimens for phylogenetic estimation. The six primer combinations could be amplified for WRT329 and WRT345, resulting in a total of 931 and 927 base pairs for these samples, respectively. For WRT349, the 5′-most-amplicon, i.e. primer combination tu157F-tu391R, could not be amplified resulting in a shorter reconstructed sequence of 771 bp (Fig. 1). WRT329 and WRT349 shared full (100%) nucleotide identity, while WRT345 exhibited one base pair variation at site 355 of the alignment (Figs 13–14), corresponding to a non-synonymous substitution at a 1st codon position. Caulerpa floridana differed from the most common haplotype of sister species C. ashmeadii (e.g. AJ417941) at eight sites of the alignment (site 240, 408, 792, 840, 858, 882 and 894, all synonymous substitutions at 3rd codon positions, and at site 764, a non-synonymous substitution at a 2nd codon position), six of which are distributed on the 3′ half of the gene tufA. Likewise, C. floridana differed from the unknown related species Caulerpa sp. from the FMG (AJ417962, Famà et al., 2002) at seven sites of the alignment (at site 687, 771, 795, 798, 858 and 894, all synonymous substitutions at the 3rd codon position, and at site 764, a non synonymous substitution at a 2nd codon position) all lying on the 3′ half of tufA. The distribution pattern of these substitutions along the stretch of tufA for the taxa investigated is contrasted in the multiple sequence alignments presented in Fig. 13 vs. Fig. 14. Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta, Bryopsidales) from the Dry Tortugas, FloridaAll authorsThomas Sauvage, Michael J. Wynne, Valerie J. Paul Suzanne Fredericqhttps://doi.org/10.1080/09670262.2014.947330Published online:04 September 2014Figs 13–14. Multiple sequence alignment of tufA detailing overlap between short amplicons generated for C. floridana specimens WRT329, WRT345 and WRT349, and sequence variation with closely related species C. ashmeadii, C. prolifera and C. sp. from the Florida Middle Ground (FMG). Conserved sites are shown as (.) in reference to C. prolifera. Fig. 13. Overlap between short amplicons tu283F-tu599R/tu459F-tu686R (position 244 to 394 of the analysed alignment in RAxML). Note site variation (Adenine: A) at position 355 (shaded in gray) for both amplicons generated for WRT345. Note low (lack) of variation on this stretch of tufA with sister species C. ashmeadii and C. sp. Fig. 14. Overlap between short amplicons tu755F-tu1017R/tu853F-tu1118R (position 719 to 870). Note nucleotide variation for C. floridana’s specimens at site 764 (Guanine: G) and 858 (Adenine: A) (shaded in grey).Display full sizeFigs 13–14. Multiple sequence alignment of tufA detailing overlap between short amplicons generated for C. floridana specimens WRT329, WRT345 and WRT349, and sequence variation with closely related species C. ashmeadii, C. prolifera and C. sp. from the Florida Middle Ground (FMG). Conserved sites are shown as (.) in reference to C. prolifera. Fig. 13. Overlap between short amplicons tu283F-tu599R/tu459F-tu686R (position 244 to 394 of the analysed alignment in RAxML). Note site variation (Adenine: A) at position 355 (shaded in gray) for both amplicons generated for WRT345. Note low (lack) of variation on this stretch of tufA with sister species C. ashmeadii and C. sp. Fig. 14. Overlap between short amplicons tu755F-tu1017R/tu853F-tu1118R (position 719 to 870). Note nucleotide variation for C. floridana’s specimens at site 764 (Guanine: G) and 858 (Adenine: A) (shaded in grey).The overall resolution of the ‘best tree’ obtained with RAxML (Stamatakis, 2006) was congruent with previous phylogenetic estimations run with either maximum likelihood or Bayesian inference (e.g. Famà et al., 2002; Stam et al., 2006). The branching order of the backbone was fully unresolved, whereas numerous clades at the tip of the tree received high support (Fig. 8). For figure clarity several clades were collapsed; these represented either single entities or multiple species-level entities, i.e. each of the lineages of the polyphyletic C. racemosa-peltata complex (A–F), and nested haplotypes of C. cupressoides among those of C. serrulata. Additional polyphyletic taxa were entities of the C. brachypus complex and the C. scalpelliformis complex. Caulerpa floridana belonged to a moderately supported grouping of species including the pantropical C. taxifolia (and var. distichophylla, Jongma et al., 2013), the Mediterranean and amphi-Atlantic C. prolifera, and the western Atlantic/Gulf of Mexico endemic C. ashmeadii from the Florida Keys and western Florida (Stam et al., 2006), and an unknown taxon labelled Caulerpa sp. from northwestern Florida (Florida Middle Grounds (FMG), Famà et al., 2002). Caulerpa floridana emerged as a sister taxon to both C. ashmeadii and the unknown Caulerpa sp. Although lying on distinct (long) branches, their topological relationship (i.e. branching order) was also moderately resolved.The morphological attributes of C. floridana were congruent with the description of Taylor (1960) for the contraction present at the base of the pinnules and a conspicuous apiculus at their top (Figs 2–6, 11). By comparison, C. ashmeadii exhibited characteristic cylindrical pinnules (no contraction) and was devoid of apiculi (Figs 7, 11). Morphological observations made on high-resolution scans of the three SPF specimens of C. floridana reported by Joly et al. (1976) from Brazil (see Fig. 9 for SPF04257; SPF04255 and SPF04256 not shown) lacked similarity with the Dry Tortugas specimens, both in their general habit and pinnule attributes. Although Joly et al. (1976) reported the presence of apiculi for these specimens, this character could not be unequivocally observed at the tip of the pinnules when zoomed in (not shown). Moreover, no contractions at the base of the pinnules were observed in the Brazilian material. Branchlets appeared cylindrical and variably positioned around the main axis of the assimilator, i.e. radial and/or distichous, a habit that was not observed in any of the 10 Dry Tortugas specimens examined for the present study (Figs 2–6, Table 2). This habit has also never been reported for C. ashmeadii, which, similar to the Joly specimens, exhibits cylindrical branchlets but no apiculi (Fig. 11). Interestingly, specimen SPF04257 exhibits two morphologically variable thalli obtained from a common dredge that Joly annotated differently doubting their identity despite their later publication as C. floridana; the right specimen is pencilled as ‘floridana?’ and the left specimen as ‘ashmeadii?’ (Fig. 9). The ‘Albatross’ specimen from Brazil annotated as ‘C. taylori Howe inedit’, i.e. C. floridana, but published as C. ashmeadii by Taylor (1930), in spite of the presence of occasional apiculi (see Taylor’s sketched arrows, Fig. 10), led to similar inconclusive identification. Overall, the identity of the southwestern Atlantic specimens recorded as C. floridana by Joly et al. (1976) could not be validated. These specimens, including the ‘Albatross’ specimen, may also differ from C. ashmeadii by the occasional presence of apiculi as reported by Joly et al. (1976) and Taylor (1930), although not clearly observed here (these are perhaps more visible on fresher material). Finally, the undescribed Caulerpa sp. sequenced by Famà et al. (2002) from the FMG lacked any pinnules consisting of filiform assimilators with no additional morphological characters (Fig. 12). In the absence of additional specimens sharing a similar sequence to assess morphological variation, no further taxonomic decision is possible at this point.DiscussionWe successfully resolved the identity and phylogeny of the enigmatic species Caulerpa floridana from the Dry Tortugas by reconstructing up to 931 bp of the gene tufA from degraded, historical DNA extracted from three 90-year-old herbarium specimens originally collected by W.R. Taylor at this locality. Sequence reconstruction was made possible by a series of newly developed Caulerpa-specific primers designed to amplify the near-complete sequence of tufA in short overlapping DNA segments, which, after assembly, allowed sufficient resolution for the molecular separation of C. floridana from the closely related species C. ashmeadii and an unknown filiform Caulerpa sp. from the FMG (Famà et al., 2002). Reconstructed sequences shared 100% nucleotide identity with the exception of WRT345, which exhibited a non-synonymous substitution at a site that is fully conserved throughout the Caulerpaceae sequenced to date for tufA (Fig. S1). Although this substitution may be endogenous, (intrinsic to the population of C. floridana rather than a PCR artefact) a miscoding lesion introduced by Taq during strand synthesis over a degraded base (Hofreiter et al., 2001; Brotherton et al. 2007; Evans, 2007; Staats et al., 2011) cannot be ruled out at this time. Miscoding lesions are often induced by the natural deamination of cytosine to uracil in preserved specimens (Brotherton et al., 2007) and most commonly represent C→T/G→A substitutions, although C→A/G→T substitutions (as observed here) are also known to occur but at a lower rate (21.4% vs. 4.5% of damage-induced mismatch in PCR-amplified historical plastid DNA extracted from herbarium specimens, respectively) (Staats et al. 2011). However, the fact that amplicons tu283F-tu599R (316 bp) and tu459F-tu686R (226 bp) (Fig. 13), and their associated chromatograms (not shown), were both unambiguous for Adenine (A), suggests this substitution is a potentially true evolutionary event. Dubious substitutions from degraded DNA samples (or even formalin-fixed specimens, Do Dobrovic, 2012) may be resolved by treating extracted templates with uracil-DNA-glycosylase (UDG) (e.g. Hofreiter et al. 2001), which cleaves DNA strands at uracil sites. Taq polymerase with proofreading activity is also strongly inhibited by uracil-containing DNA and may thus be preferred to prevent extension over mutated sites (Lasken et al., 1996). Here, considering the minor impact of this single substitution on the phylogeny of C. floridana (and non-ambiguous chromatograms), we did not attempt UDG treatment. Lastly, the multiple sequence alignments displayed in Figs 13–14 indicate that substitutions among C. floridana and sister species were mostly distributed on the 3′ half of tufA. From this practical observation, generating sequence data for amplicons tu585F-tu818R, tu755F-tu1017R and tu853F-tu1118R (eventually in a multiplex way, e.g. Alonso et al., 2003) may thus be prioritized for the molecular confirmation of historical specimens suspected to belong to this clade, significantly reducing manipulation time and associated costs.The specimens reported as C. floridana by Joly et al. (1976) in the southwestern Atlantic, off the States of Ceará and Bahia in Brazil, could not be confirmed as conspecifics based on their overall habit (Figs 2–6 vs. Fig. 9) and especially the lack of conspicuous apiculi, although reported by these authors. It appears that Joly et al. (1976) doubted the identity of these specimens as seen from the notes pencilled on the two sheets of SPF04257, ‘floridana?’ and ‘ashmeadii?’ (Fig. 9). It is interesting that the identification of the ‘Albatross’ specimen (T13894) as C. ashmeadii from the State of Pernambuco, Brazil (Fig. 10), was also a source of confusion prior to its publication by Taylor (1930). Taylor followed the opinion of M.A. Howe for its identification (see annotation ‘verif. M.A. Howe, Oct. 1929’), despite the observation of apiculi that he thought would place the specimen in C. floridana, as seen from his annotation ‘But: habit is that of the segregate \"C. Taylori Howe inedit.” and there are traces of apiculi.’ that he pointed at with an arrow (Fig. 10). Clearly, the identity of the Brazilian specimens is quite equivocal and they cannot be attributed to either C. floridana or C. ashmeadii at this point. Further work is needed to shed light on the Brazilian entities. In this endeavour, the molecular identity of specimens previously reported as C. ashmeadii from the Antillean arc in the Caribbean should also be investigated for comparison and to further define the southward distribution of this species. It should be noted that some distichous Caulerpa specimens displaying somewhat cylindrical to clavate pinnules reported as C. racemosa var. lamourouxii in the Western Atlantic (Caribbean Panama: Wysor Kooistra, 2003; Bermuda: Schneider Lane, 2007), may represent individuals from Lineage A of the C. racemosa-peltata complex (sensu Sauvage et al., 2013), corresponding to C. chemnitzia (Esper) J.V. Lamour. in a recent taxonomic revision of the complex by Belton et al. (2014), which could potentially be misidentified as C. ashmeadii elsewhere in the basin. Likewise, the report of C. ashmeadii from Vietnam by Tien (2007, cited in Guiry Guiry, 2014) most probably represents a separate lineage. The lack of a confirmed record of C. floridana in the western Atlantic other than from the Dry Tortugas (White Shoal and Southwest Channel, Table 2) is curious and calls for further exploration in the region, especially from deeper environments of the Gulf of Mexico and vicinity ( 18 m depth).Caulerpa floridana was a sister species to both C. ashmeadii and an unknown filiform Caulerpa sp. from the FMG (western Florida), all of which are endemic to the western Atlantic to our knowledge. Interestingly, this radiation shared a common ancestor with C. prolifera, a species that is also restricted geographically with an amphi-Atlantic and Mediterranean Sea distribution, thus further supporting the hypothesis of a local diversification of these endemics in the western Atlantic, and possibly in the Gulf of Mexico. Herbivory assays conducted on C. ashmeadii by Paul et al. (1987) demonstrated its strong avoidance by grazers in comparison to other Caulerpa species, including C. prolifera. Chemical investigation of C. ashmeadii revealed four new cyclic sesquiterpenoid metabolites, which are unique among the Caulerpaceae, present in high concentration in contrast to the linear sesquiterpene caulerpenyne, which is present in C. prolifera and many other Caulerpa species (Paul Fenical, 1986). These secondary metabolites appeared to explain the resistance of C. ashmeadii to grazers by acting as herbivore deterrents for chemical defence (Paul et al., 1987). However, it is currently unknown whether these metabolites are also biosynthesized in the closely related sister species C. floridana and the FMG Caulerpa sp., i.e. whether these sesquiterpenoids represent an autapomorphic trait in C. ashmeadii or a synapomorphy among these three species. Chemical defence through the production of potent sesquiterpenoids could represent an adaptive trait (Doebeli, 2011), which may have played a role in the diversification of the lineage in the western Atlantic. Future studies could attempt to re-collect fresh material of C. floridana and the FMG Caulerpa sp. to conduct similar chemical analyses and examine further the above hypothesis. In addition, efforts to determine the timing of diversification in this radiation with molecular clock analyses on a better resolved phylogenetic framework (i.e. its backbone) may provide important clues toward understanding the intricacies of local/global events, which may have also contributed to the speciation of these western Atlantic endemics. Age estimates for the Caulerpacean and related Bryopsidalean lineages are available in the multi-marker study of Verbruggen et al. (2009) to calibrate such phylogeny.Finally, taking into account the apparent rarity of C. floridana in the field, its presence in the aquarium trade and risks of its release in California’s coastal waters are probably low. For instance, in their DNA barcoding survey of Caulerpa species present in the California and Florida aquarium trade, Stam et al. (2006) did not produce any tufA sequences matching those obtained here for C. floridana, while they identified C. ashmeadii in one store in California (sample CA007, DQ652362). The latter is more common and accessible in shallow waters of Florida, thus its entry in the trade may be more frequent. It is to be noted that no regulations currently exist regarding the possession and sale of Caulerpa species in Florida, other than transporting C. taxifolia across state lines (as for any other state) because of its inclusion in the Federal Noxious Weed List. This is worrisome considering that suitable habitats for foreign (alien) Caulerpa species abound on the coastline of Florida inhabited by 10 native taxa (personal observations), some remaining to be clarified molecularly and taxonomically (e.g. blooms of C. brachypus f. parvifolia (Harvey) A.B. Cribb, Lapointe et al., 2005). Recently, Diaz et al. (2012) demonstrated the ineffectiveness of the ban raised in California in 2001 on the possession or sale of C. taxifolia and other designated Caulerpa species (see listing in introduction) in this state, suggesting that regulations, even when existing, are difficult to enforce for these organisms. Nonetheless, according to the Precautionary Principle (Cooney, 2004), Diaz et al. (2012) supported the retention of the ban. Awaiting further clarification of Caulerpacean taxa found around the state of Florida, we hope that our morphological illustrations of C. floridana confirmed by DNA sequencing of historical material will facilitate identification of this beautiful species in the field, museum herbarium collections and potentially in the aquarium trade.Supplementary informationThe following supplemental material is available for this article, accessible via the Supplementary Content tab on the article’s online page at http://dx.doi.org/10.1080/09670262.2014.947330Fig. S1. Character variation in a 63 bp stretch of the chloroplast protein-coding gene tufA including site 355 (highlighted in a light grey column). Note the substitution for WRT345/KF921072 (top sequence) for Adenosine (A) where the remainder of the Caulerpaceaeae is fully conserved for Cytosine (C), including WRT349/KF921073 (second sequence from the top) for comparison. The figure was produced through exhaustive BLAST searches of 800 bp Caulerpa spp. queries on NCBI GenBank followed by dereplication at the 63 bp stretch of interest.Table 1. Newly designed primers for the present study with position based on the Ostreococcus tauri Courtes Crétiennot complete tufA sequence (CR954199). GC: Guanine-Cytosine content; Tm: melting temperature; bp: primer length in base pairs.NameSequencebpGC%Tm (°C)tu157F15′- GCWCCWGAAGAAAAAGC - 3′1747.148.6tu283F5′- GGAGCAGCTCAAATGG -3′1656.350.5tu459F5′- AATACGAGAAACATTAG - 3′1729.439.9tu585F5′- GGTGGATAAAATTTATC - 3′1729.438.7tu755F5′- CTGTGGAAGTTATTG - 3′1540.039.6tu853F5′- GTTGGTATTCTTTTAC - 3′1631.337.8tu391R5′- TAGCAGGAACGCC - 3′1361.547.1tu599R5′- TAAATTTTATCCACCCAAG-3′1931.644.6tu686R5′- ACAACATTTTCNACAG - 3′1634.442.0tu818R5′- TGAAACATTTCTAGACC - 3′1735.342.8tu1017R5′- AGGTCTATACCCAG - 3′1450.040.8tu1118R5′- TTCACTCGATCTCC - 3′1450.042.61 Previously published in Sauvage et al. (2013).Table 2. Herbarium specimens of C. floridana sensu stricto present in MICH and examined in the present study. All originally collected from the Dry Tortugas, White Shoal, and off Southwest Channel.SpecimenSta.LocationDepthDateCollector––––07/–/1915H.H. BowmanWRT28726White Shoal18 m7/19/1924W.R. TaylorWRT30933White Shoal–7/10/1924W.R. TaylorWRT329138White Shoal73 m7/21/1924W.R. TaylorWRT3451,239White Shoal91 m–/–/1924W.R. TaylorWRT349140White Shoal109m7/22/1924W.R. TaylorWRT361319White Shoal21 m7/17/1924W.R. TaylorWRT38851White Shoal––/–/1924W.R. TaylorWRT692214off SW Channel29 m6/11/1925W.R. TaylorWRT663211off SW Channel18 m6/11/1925W.R. TaylorWRT1393330off SW Channel34 m6/20/1926W.R. Taylor1 Specimens selected for molecular assessment, see Figs 4–6.2 Mounted on the bottom half of the Holotype herbarium sheet.3 Holotype (cross referenced MICH1306213) and Isotype (cross referenced NY01027398), see Figs 2–3.Table 3. Southwestern Atlantic specimens from Brazil reported as C. floridana or C. ashmeadii by Joly et al. (1976) and Taylor (1930).SpecimenStationStateSiteDepthDateSPF042571,2E1688CearáAlm. Saldanha49 m10/10/1967SPF042561D-9BahiaAlm. Saldanha47 m9/26/1967SPF042551E1729ACearáAlm. Saldanha35 m10/30/1967T1389432758Pernambucona36 m12/16/18871 Maintained at Universidade de São Paulo herbarium (SPF).2 SPF04257 harbours two morphologically variable thalli originating from a common collection (see Fig. 9).3 Maintained at University of Michigan herbarium (MICH).Supplemental materialSupplementary materialDownload PDF (105 KB)AcknowledgementsTS is indebted to the Link Foundation/Smithsonian Marine Station 2013 Fellowship Award for funding residency in Florida and at SMS for the completion of this research. TS is thankful to Rahul Ukey and Drs A. Chistoserdov and W. Schmidt (UL Lafayette), and Drs M. Boyle, N. Engene and J. Sneed (SMS) for technical and/or field assistance. Dr Ken Karol at NY is thanked for uploading an image of the isotype of Caulerpa floridana to the C.V. Starr Virtual Herbarium. 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