Phycological Research 2010; 58: 69–77 Chamaebotrys prolifera sp. nov. (Rhodymeniaceae, Rhodophyta) from Puerto Rico, Caribbean Sea, based on morphology and small subunit rDNA sequences pre_560 69..77 David L. Ballantine,* Hector Ruiz and Chad Lozada-Troche Department of Marine Sciences, P.O. Box 9013, University of Puerto Rico. Mayagüez, Puerto Rico 00681 SUMMARY A new rhodymeniacean species, Chamaebotrys prolifera, is described from a shallow water habitat in Puerto Rico, representing the first occurrence of the genus in the Atlantic Ocean. Plants, to 5 cm across, are decumbent and comprised of compressed vesicles that are originally proliferously branched at the perimeter. Older vesicles become branched from their dorsal surfaces as well. Branches are septate at their origin and become irregularly shaped with age. Anastomoses between adjacent vesicles is common. Individual vesicles measure to 15 mm in broadest dimension. Vesicle walls consist of two layers of medullary cells and two layers of cortical cells. Tetrasporangia, which occur in diffuse nemathecia, are spherical, to 30 mm in diameter and are cut off terminally from an inner cortical cell. Cystocarps are hemispherical measuring to 800 mm in diameter and 350 mm in height. Spermatangia are apparently cut off randomly from outer cortical cells across the thallus surface. Molecular evidence confirms placement of Chamaebotrys within the Rhodymeniaceae. Key words: Chamaebotrys prolifera sp. nov., Puerto Rico, Rhodophyta, Rhodymeniaceae, small subunit rDNA. INTRODUCTION Chamaebotrys was segregated from Coelarthrum Børgesen by Huisman (1996) with Chamaebotrys boergesenii (Weber Bosse) Huisman designated as the generitype. Chamaebotrys boergesenii differs from Coelarthrum on the basis of habit, that is, decumbent plants attached at several points, commonly possessing secondary anastomoses, and reproductively in the formation of tetrasporangia in a terminal rather than in an intercalary position within nemathecia (Huisman 1996). The transfer of a second species also formerly treated as Coelarthrum, C. lomentariae (Tanaka & K.Nozawa) Huisman was also made at that time. A third species, Chamaebotrys erectus Schils & © 2010 Japanese Society of Phycology Huisman, was recently established by Schils et al. (2003). The erect habit of this later species broadened the generic circumscription somewhat. Currently all Chamaebotrys species are known from the Pacific and Indian Oceans: C. boergesenii is widely distributed in the Indo-Pacific (Huisman 1996), C. erectus is known only from its type locality, Yemen, Indian Ocean (Schils et al. 2003) and C. lomentariae is endemic to Japan (Tanaka 1964; Yoshida et al. 1990). Continuing study of the marine algal flora of Puerto Rico has resulted in recognition of a new species of Chamaebotrys, which represents the first occurrence of the genus in the Atlantic Ocean. MATERIALS AND METHODS Specimens were collected by snorkeling and then either preserved in 10% Formalin/seawater or placed in silica gel in a glass container. Transections (30–40 mm thick) were made with an American Optical Cryo-Cut freezing microtome (Leica, Wetzlaar, Germany). Microscope slide preparations were stained with 1% acidified aniline blue and mounted in 60% Karo syrup. Photomicrographs were taken with a Spot RE digital camera through an Olympus BMAX light microscope. The plates were assembled from digital photographs utilizing Adobe Photoshop CS2. Voucher specimens have been deposited in MICH, MSM and US. Herbarium abbreviations follow Holmgren et al. (1990), and authority designations are in accordance with Brummitt and Powell (1992). Molecular analyses used the small subunit (SSU rDNA) and RUBISCO (rbcL) genes. DNA extraction, polymerase chain reaction (PCR) amplification and sequencing were carried out using the protocols described in Ballantine and Lozada-Troche (2008). Phylogenetic reconstruction was carried out using the *To whom correspondence should be addressed. Email: david.ballantine@upr.edu Communicating editor: O. DE Clerck. Received 14 November 2008; accepted 13 June 2009. doi: 10.1111/j.1440-1835.2009.00560.x 70 D. L. Ballantine et al. maximum parsimony (MP), neighbor joining (NJ) and maximum likelihood (ML) algorithms as implemented in PAUP* (Swofford 2002) and Bayesian Inference using MrBayes (Huelsenbeck & Ronquist 2001). Modeltest (Posada & Crandall 1998) was used to determine the correct DNA substitution model to be used as input for NJ and ML analyses and the Bayesian Information Criterion for Bayesian Inference. The optimal model found for the SSU rDNA analysis was the GTR + I + G evolutionary model (General Time Reversal model + Invariable Sites + Gamma Distribution) (Lanave et al. 1984; Rodriguez et al. 1990). Assumed nucleotide frequencies were: A = 0.2427, C = 0.2126, G = 0.2850, T = 0.2597. The assumed substitution rate matrix was: A-C = 0.9958, A-G = 3.1979, A-T = 1.2154, C-G = 0.5727, C-T = 4.2088, G-T = 1.0000. Proportion of sites assumed to be invariable = 0.6941; rates for variable sites were assumed to follow gamma distribution with shape parameter = 0.8019. Bayesian Inference was done using the K80 + I + G evolutionary model (Kimura 2-parameter + Invariable sites + Gamma Distribution) (Kimura 1980). Assumed nucleotide frequencies were equal for all bases. The proportion of sites assumed to be invariable = 0.6956; rates for variable sites were assumed to follow a gamma distribution with shape parameter = 0.7912. Table 1. The optimal model for rbcL data also was: (GTR + I + G). Assumed nucleotide frequencies were: A = 0.3043, C = 0.1515, G = 0.2248, T = 0.3195. The assumed substitution rate matrix was: A-C substitutions = 1.4593, A-G = 6.2946, A-T = 4.0647, C-G = 1.6844, C-T = 18.0794, G-T = 1.0000; proportion sites assumed to be invariable = 0.5390; rates for variable sites were assumed to follow a gamma distribution with shape parameter = 2.7617. Bayesian analysis of rbcL sequences was done following the TIM + G evolutionary model (Transition model + Gamma Distribution) (Posada & Crandall 2001). The robustness of 18S and rbcL data was determined by bootstrapping the dataset (Felsenstein 1985) 2000 times for MP and NJ and 1000 times for ML. Bayesian analysis was conducted running 1 000 000 generations. Trees were sampled every 100 generations with log-likelihood scores stabilized at approximately 2500 generations. For 18S sequence analysis the first 3000 trees of a possible 10 000 trees were discarded as burn-in. Analysis of rbcL sequences stabilized at 3500 generations and the first 4000 trees were discarded as burn-in. Tables 1 and 2 show species used (and source) for the SSU rDNA and rbcL gene sequences, respectively. Results from the newly sequenced species are deposited in GenBank (Table 1, accession numbers in bold). List of species used in the18S (small subunit [SSU]) gene sequence analysis (numbers in bold are new to GenBank) Species Source Accession Number Reference Chamaebotrys prolifera (DLB-7257) Chamaebotrys prolifera (DLB-7257) Coelarthrum cliftonii (CLT-197) Coelarthrum cliftonii (DLB-7260) Coelarthrum cliftonii (DLB-7260) Coelarthrum opuntia Chrysymenia agardhii (DLB-6000) Chrysymenia nodulosa (CLT-181) Chrysymenia ornata Erythrocolon podagricum Gloiosaccion brownii Halymenia plana Sebdenia flabellata San Juan, Puerto Rico San Juan, Puerto Rico Guanica, Puerto Rico Guanica, Puerto Rico Guanica, Puerto Rico GenBank La Parguera, Puerto Rico La Parguera, Puerto Rico GenBank GenBank GenBank GenBank GenBank EU715131 EU715132 EU086465 EU670594 EU670595 AF085258 EF690262 EF690261 AF085257 U23953 AF085269 U33133 U33128 This paper This paper This paper This paper This paper Saunders et al. 1999 This paper This paper Saunders et al. 1999 Millar et al. 1996 Saunders et al. 1999 Saunders & Kraft 1996 Saunders & Kraft 1996 Table 2. List of species used in the rbcL gene sequence analysis. (numbers in bold are new to GenBank) Species Source Accession Number Reference Chamaebotrys prolifera (DLB-7257) Chamaebotrys prolifera (DLB-7257) Coelarthrum cliftonii (DLB-7260) Chrysymenia agardhii (DLB-6000) Chrysymenia procumbens Halymenia floresii Sebdenia integra San Juan, Puerto Rico San Juan, Puerto Rico Guanica, Puerto Rico La Parguera, Puerto Rico GenBank GenBank GenBank EU715134 EU715135 EU715136 EU715133 AY294381 AB038603 AY294363 This paper This paper This paper This paper Gavio et al. 2005 Wang et al. 2000 Gavio et al. 2005 © 2010 Japanese Society of Phycology A new Chamaebotrys species from Puerto Rico RESULTS Chamaebotrys prolifera sp. nov. Plantae decumbentes, ad sitos numerosos brevibus hapteronibus paxilliformibus affixi; coloniae integrae usque ad 5.0 cm latae, e vesiculis compressis et ovatis usque ad irregularibus constantes, usque ad 15 mm ut maximum; ramificatio prolificans a margine, non plerumque e superficiebus dorsalibus ramose; rami septati; vesiculae contiguae plerumque anastomosantes; paries vesiculae plerumque crassus 4-cellulas; stratum medullosum, quod crassum 2-cellulas est, e strato interiore magnarum cellularum rectangularium constans, ut visum in sectione 125– 165 (-200) ¥ 65–100 mm, atque vel e strato partiali cellularum triangularium medullosarumque conferto inter cellulas medullosas maiores usque ad 50–75 mm ut maximum, vel e strato continuo localique cellularum medullosarum minorum; cortex, ex 2 stratis cellularum constans, stratum interiorem cellularum sphaericarum usque ad ovatas, 15–20 mm lato, praebens; stratus extimus cellularum 5.0–7.5 mm diametro; cortex cellulas medullsas omnino tegens; glandicellulae numerosae (2 ad 12) et pyriformes, usque ad 17.5 mm longae ¥15 mm diametro, a magnis cellulis ferentibus et incoloratis abscissae quae in interioribus cellulis medullosis portata sunt aut rarius directe a interioribus cellulis medullosis; tetrasporangia sphaerica, usque ad 30 mm diametro, a interiore cellula corticali terminaliter abscissa et in nematheciis diffusis locata; tetrasporangia paraphysibus unicellularibus circumcincta; plantae monoeciae; cystocarpia hemisphaerica, usque ad 800 mm diametro et 350 mm alta, intrinsecus et extrinsecus aeque protrudentia; pauca spermatangia abscissa, ut videtur fortuito a exterioribus cellulis corticalibus trans superficiem thalli. Plants decumbent, attached at numerous sites with short (to 1.0 mm long) peg-like holdfasts; entire colonies to 5.0 cm across; comprised of compressed ovate to irregularly-shaped vesicles, measuring to 15 mm in largest dimension, with proliferous branching from margins, less commonly branched from the dorsal surfaces; branches septate; adjacent vesicles commonly anastomosing; vesicle wall generally four cell layers thick, the two cell-thick medullary layer consisting of an inner layer of large rectangular cells, when viewed in section measuring 125–165 (-200) ¥ 65–100 mm, and either a partial layer of triangular medullary cells wedged between the larger medullary cells, measuring 50–75 mm in broadest dimension or a locally continuous layer of smaller medullary cells; the two cell-layer cortex with an inner layer of spherical to oval cells, 15–20 mm in broadest dimension, the outermost layer of spherical cells measuring 5.0–7.5 mm in diameter; the cortex © 2010 Japanese Society of Phycology 71 completely covering the medullary cells; numerous (2–12) pyriform gland cells, to 17.5 mm long and 15 mm in diameter, cut off from large colorless bearing cells that are borne on inner medullary cells or more rarely, directly from the inner medullary cells; tetrasporangia spherical, to 30 mm in diameter cut off terminally from an inner cortical cell and located in diffuse nemathecia and surrounded by single-celled paraphyses; plants monoecious, cystocarps hemispherical measuring to 800 mm in diameter and 350 mm in height; and protruding equally inwardly and outwardly; spermatangia cut off in few numbers, apparently randomly from outer cortical cells across the thallus surface. Holotype. D.L. Ballantine 7257, San Juan Bay (Pier 8), San Juan, Puerto Rico (18°27.766′N, 66°06.202′W), 1–2 m, coll. H. Ruiz, 4.xi.2007 (Alg. Coll. # US-209253). Isotypes: MICH: MSM; US. Paratypes. D.L.B. 7211, ibid, 6.x.2007; D.L.B. 7443, ibid, 11.ii.2008. Etymology. The specific epithet refers to the proliferous generation of new segments from parent vesicles. Plants originate as single compressed, peltate vesicles, being attached by one or more peg-like holdfasts. The primary vesicles measure to 15 mm in diameter and are typically ovate. Originally branches are produced from the margins (Figs 3,4) with young branchlets being ovate-compressed. The branchlets enlarge and similarly rebranch (Fig. 5). With age, branches may also form on the dorsal surface with an increasing number of vesicles being irregular in shape (Figs 5,6). Branches are septate at their origin (Figs 3– 5,11). Lateral attachments commonly occur between adjacent vesicles (Figs 4–6). Mature plants are a chaotic arrangement of vesicles (Figs 1,2,6) with the entire plant measuring to 5.0 cm broad. Older vesicles may tear with the wall remaining viable and supporting continuing branching (Fig. 7). The vesicle walls possess two layers of colorless medullary cells and two layers of cortical cells (Figs 8–10). The innermost medullary cells are rectangular and measure 125–165(-200) ¥ 65– 100 mm. Between the inner medulla and the cortex is a partial layer of triangular medullary cells wedged between the larger medullary cells (Fig. 8), measuring 50–75 mm in broadest dimension or a locally continuous layer of smaller medullary cells. The two-cell layer cortex consists of an inner layer of spherical to oval cells, 15–20 mm in broadest dimension. The outermost cortical cells are spherical and measure 5.0–7.5 mm in diameter. The cortex completely covers the medullary cells. Numerous (to 12) pyriform gland cells, to 17.5 mm long by to 15 mm in diameter are cut off from large colorless bearing cells that are borne on inner medullary cells (Fig. 9) or more rarely directly from the inner medullary cells (Fig. 10). The septa consist of multilayered medullary cells that increase in layer number with age (Fig. 11). 72 Figs 1–7. D. L. Ballantine et al. Chamaebotrys prolifera sp. nov. (all DLB-7257). 1. Habit of large specimen showing multiple irregularly arranged vesicular branches. Scale bar, 1.0 cm. 2. Habit of the holotype. Scale bar, 1.0 cm. 3. Primary vesicle with marginal vesicular branches. Scale bar, 2.0 mm. 4. Primary vesicle with marginal branches, many of which are laterally connected (arrowheads) and a dorsally placed branch (asterisk). The plant is cystocarpic (arrows). Scale bar, 2.0 mm. 5. Plant in which the primary branches are becoming irregular in shape and giving rise to a new order of branchlets. Arrowhead denotes lateral fusion. Scale bar, 5.0 mm. 6. Close up of irregularly shaped branches bearing cystocarps (arrows). Lateral fusions are denoted with arrowheads. Scale bar, 5.0 mm. 7 Portion of vesicle that has split open, but remains viable. Scale bar, 1.0 cm. Tetrasporangia are spherical, to 30 mm in diameter, and are cut off terminally from an inner cortical cell (Fig. 12) and are located in somewhat diffuse sori. Adjacent to the tetrasporangia-producing cortical cells, cortical cells also produce single-celled sterile paraphyses (Fig. 12). Plants are monoecious. Cystocarps are hemispherical measuring to 800 mm in diameter and 350 mm in height; they protrude equally inwardly and outwardly (Fig. 13). Spermatangia are cut off in few numbers, apparently randomly from outer cortical cells across the thallus surface. © 2010 Japanese Society of Phycology A new Chamaebotrys species from Puerto Rico 73 Figs 8–13. Chamaebotrys prolifera sp. nov. 8. Cross-section through vesicle wall, showing two cell-layered medulla (DLB-7257). Scale bar, 100 mm. 9. Cross-section through vesicle wall showing an accessory medullary cell bearing gland cells (DLB-7257). Scale bar, 100 mm. 10. Cross-section through vesicle wall, in which regular medullary cells give rise to small gland cells (DLB-7257). Scale bar, 100 mm. 11. Cross-section through vesicle wall at site of multicellular septum at point of branching (DLB-7257). Scale bar, 250 mm. 12. Cross-section through vesicle wall showing terminally placed tetrasporangia (arrows) (DLB-7443) with adjacent paraphyses. Scale bar, 50 mm. 13. Cross-section through cystocarp (DLB-7257). Scale bar, 100 mm. Lengths of new SSU rDNA sequences obtained from Chamaebotrys prolifera and other Rhodymeniaceae species varied from 1697 to 1756 bp. A maximum likelihood tree inferred from the SSU rDNA sequence data (Fig. 14) indicates that C. prolifera is a sister taxon of the genera Coelarthrum and Erythroco© 2010 Japanese Society of Phycology lon. Approximately 1291 bp of the Rubisco (rbcL) gene were also newly sequenced for specimens of C. prolifera, Coelarthrum cliftonii (Harv.) Kylin and Chrysymenia agardhii Harv. A maximum likelihood tree inferred from rbcL sequence data (not shown) also indicates a highly supported separation between 74 D. L. Ballantine et al. Fig. 14. Small subunit (SSU) (18s) sequence phylogram using maximum likelihood. Bootstrap proportions are shown on top of the branches, left to right: Bayesian inference (1 000 000 generations), maximum parsimony (2000 replicates), neighbor joining (2000 replicates), and maximum likelihood (100 replicates). Chamaebotrys prolifera and Coelarthrum cliftonii with an 11% divergence between them and are supportive of the taxonomic relationship revealed by the SSU rDNA analysis. DISCUSSION Huisman (1996) presented a table of differentiating characters for hollow rhodymeniacean genera. Four of these: Chamaebotrys, Coelarthrum, Erythrocolon and Webevanbossea, as is the new species, are regularly segmented. Coelarthrum and Erythrocolon differ from the new species by possessing tetrasporangia that originate in intercalary positions (Huisman 1996). Erythrocolon and Webervanbossea produce gland cells borne on internal filaments (Huisman 1995) that differ from gland cell origin in the new entity. Webervanbossea, however, is now placed in the Faucheaceae (Saunders et al. 1999; Schneider & Wynne, 2007). On these bases, the new species would appear to be most closely related to Chamaebotrys. Unfortunately, there are no prior molecular data for Chaemobotrys with which to compare the sequences obtained of the new species. Nevertheless, the new species does closely group with Erythrocolon and Coelarthrum, the genus from which Chamaebotrys was segregated. Chamaebotrys is currently constituted by species (Table 3) ‘similar in morphology to Coelarthrum but differing in tetrasporangial initiation’ (Huisman 1996, p. 108). Chamaebotrys prolifera differs from all other members of the genus in lacking a strong resemblance to Coelarthrum. Nevertheless Wynne (2001) figured two highly irregular thalli of putative Chamaebotrys boergesennii f. minimus (WeberBosse) M.J. Wynne, neither with gross morphological similarity to Coelarthrum. Chamaebotrys prolifera further differs from C. boergesenii on the basis of its chiefly polychotomous branching as opposed to mostly © 2010 Japanese Society of Phycology This paper Narrow On elongate nemathecial filaments, 20–25 ¥ 27–40 To 15 ¥ 17.5 mm To 30 mm © 2010 Japanese Society of Phycology W ¥ L, width ¥ length. Decumbent, To 15 mm colony to 5 cm Puerto Rico Chamaebotrys prolifera Polychotomous, proliferous Present 2 6.5–20.5 2 Erect. 20 cm high 7 ¥ 20 mm Yemen, Indian Ocean Chamaebotrys erectus Dichotomous or Absent verticillate 1 Erect?, 3–5 cm To 2.5 ¥ To 6 mm Dichotomous, irregular Japan Chamaebotrys lomentariae Unknown Narrow Tanaka & Nozawa (1963) in Tanaka (1964); Yoshida et al. 1990 Schils et al. 2003 Broad 15–20 ¥ 10–15 mm Huisman 1996 20–30 ¥ 35–40 Broad Spherical, 20–25 mm diameter Spherical or ovate 1 Present Indian, Pacific Decumbent, 2–6 ¥ 2–5 mm Dichotomous, Oceans colony to 5 cm trichotomous Chamaebotrys boergesenii Habit Segment size Branching Distribution Table 3. Comparative features of Chamaebotrys species 75 Species Gland cells Number Secondary medullary lateral anastomoses layers Tetrasporangia size (mm, W ¥ L) Constrictions Reference between segments A new Chamaebotrys species from Puerto Rico di-trichotomous branching and smaller tetrasporangia (to 30 mm diameter vs. 20–30 ¥ 35–40 mm) in the latter (Huisman 1996). Chamaebotrys erectus as the specific epithet indicates, is an erect species. It otherwise differs from C. prolifera by its substantially larger size (to 20 cm high), dichotomous branching and lack of secondary anastomoses (Schils et al. 2003). Furthermore, Chamaebotrys erectus produces elongate nemathecial filaments that arise from outer cortical cells and ultimately produce tetrasporangia terminally. These elongate nemathecial filaments are not produced by either the new species or the generitype, C. boergesenii, although short single celled paraphysal-like filaments surround sporangia in C. prolifera (Fig. 12). Chamaebotrys lomentariae is a relatively poorly known entity that may prove to be conspecific with C. boergesenii (Huisman 1996), differing by its erect habit, although that character was questioned by Huisman (1996). Nevertheless, Masuda et al. (2001) emphatically regarded the two taxa as separate species. Chamaebotrys lomentariae also differs from the new species in that the constrictions between segments are barely noticeable as opposed to being distinct in the new species. In our phylogenetic tree (Fig. 14), the clade including Coelarthrum, Chamaebotrys and Erythrocolon is distinct from that containing Chrysymenia. While the generic sampling is admittedly low, both clades contain genera that produce intercalary sporangia (Chrysymenia and Coelarthrum). The obvious morphological feature that separates them is the presence of segmentation in the Coelarthrum/Chamaebotrys clade and its lack in Chrysymenia. Lee (1978) regarded genera with terminal cruciately divided tetrasporangia as belonging to Rhodymeniaceae, while Saunders et al. (1999) placed these genera in the Faucheaceae and considered that algae with intercalary cruciate tetrasporangia belong in the Rhodymeniaceae. Nevertheless, Saunders et al. (1999) speculated on the familial placement of Chamaebotrys, and despite its seeming affinity with the Facuheaceae, provisionally assigned the genus (and correctly in our opinion) to the Rhodymeniaceae. Our molecular evidence clearly demonstrates that Chamaebotrys is a member of the Rhodymeniaceae. While substantial strides have been realized in understanding Rhodymeniales taxonomy including the recent recognition of two new families (Le Gall et al. 2008), systematic difficulties within the order have been well documented (Saunders & Kraft 1994, Huisman 1995, 1996; Saunders 2004). One confounding issue in Rhodymeniaceae circumscription remains the fact that many characters are shared between genetically distinct groups. This is may be attributable to a highly conserved veg- 76 etative evolution that is illustrated by the many rhodymeniacean genera that possess essentially identical vesicle walls (Huisman 1996) as well as difficulty in recognition of evolutionary reversions (Saunders et al. 1999). ACKNOWLEDGMENTS The Sequencing and Genotyping facility, University of Puerto Rico-Río Piedras is supported in part by NCRRAABRE Grant #P20 RR16470, NIH-SCORE Grant #S06GM08102, University of Puerto Rico Biology Department, NSF-CREST Grant #0206200. We thank Sr. Angel García who facilitated permits and collection in the San Juan Bay port area. Drs Craig Schneider and John Huisman provided constructive criticism, which improved the manuscript. Ms Angela Piper provided the Latin translation. REFERENCES Ballantine, D. L. and Lozada-Troche, C. 2008. 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