Phycologia (2008) Volume 47 (2), 225—235 Published 12 M arch 2008

Systematics and biogeography of the Pseudocodiumgenus (, ), including the descriptionP. of natalense sp. nov. from South Africa

O l i v i e r D e C l e r c k 1*, H e r o e n V e r b r u g g e n 1, J o h n M . H u i s m a n 2, E t i e n n e J . F a y e 3, F r e d e r i k L e l i a e r t 1, T o m S c h i l s 11' and Eric Coppejans1

1 Phycology Research Group ancl Centre for Molecular Phylogenetics and Evolution, Biology Department, Ghent UniversityKrijgslaan 281 (S8), 9000 Ghent, Belgium 2School o f Biologiccd Sciences and Biotechnology, Murdoch University, Murdoch WA 6150, & Western Australian Herbarium, Department o f Environment and ConservationGeorge St, Kensington WA 6151, Australia 3Marine Environment and Engineering Department, Coasted and Estuarine Environment Group, Port and Airport Research Institutei-l-l Nagase, Yokosuka, 239-0826 Japan

O . D e C l e r c k , H . V e r b r u g g e n , J . M . H u i s m a n , E .J . F a y e , F . L e l i a e r t , T . S c h i l s a n d E . C o p p e j a n s . 2008. Systematics and biogeography of the genus Pseudocodium (Bryopsidales, Chlorophyta), including the description of P. natalense sp. nov. from South Africa. Phycologia 47: 225-235. D O I : 10.2216/07-79.1

This study examines the diversity of Pseudocodium, a small green algal genus assigned to its own family in the order Bryopsidales. The included species are characterized by uncalcified thalli with a medulla composed of a limited number of interwoven siphons that form a cortex of distinctive utricles toward the periphery. Detailed morphological descriptions are provided for all known species. A combination of characters relating to general habit, morphology of the axes, apices and utricles distinguishes all species easily. Re-examination of Indonesian and Papua New Guinean specimens, previously reported as Pseudocodium floridanum, reveals that they belong to the recently described Pseudocodium okinawense. South African specimens formerly attributed to P. floridanum are described as a new species, Pseudocodium, natalense. Pseudocodium australasicum, a species endemic to southern Australia, does not fit the generic concept of Pseudocodium in that it does not possess a cortex composed of utricles. Putative reproductive structures have thus far only been reported for P. floridanum. Observations on Pseudocodium devriesii and P. natalense reveal very similar spherical to irregular structures formed laterally on medullary siphons in the axils of peripheral utricles. Interestingly, and confirming the earlier observations of Dawes & Mathieson, no basal cross wall is formed at the base of the reproductive structures. Instead, a translucent plug at the base of the reproductive structures prevents the cytoplasm from leaking. This suggests that Pseudocodium is not holocarpic, unlike most Halimedineae. DNA sequences of i-Zic L and tufA are presented for all species and the resulting phylogenies are used as a framework to study the evolution of morphological features as well as the biogeography of the genus, both at the global scale and within the Indian Ocean.

K e y W o r d s : Bryopsidales, Biogeography, Indian Ocean, Pseudocodium, /-è c L , Systematics, Phylogeny, tufA

INTRODUCTION The taxonomic position of Pseudocodium within the Bryopsidales has been considered uncertain for a long time. The green alga Pseudocodium Weber-van Bosse (1896) is a On the basis of similarities in cell wall structure (Hillis- small genus assigned to its own family, the Pseudocodiaceae, Colinvaux 1984) and overall morphology (Vroom et cd. in the order Bryopsidales. The included species are 1998), Pseudocodium was placed in the suborder Bryopsi- characterized by uncalcified thalli with a medulla composed dineae, allied with Bryopsis, Codium and Derbesia. Feld- of a limited number of interwoven siphons that, toward the mann (1946), however, hypothesized that Pseudocodium periphery, form a cortex of distinctive, laterally coherent would be more closely related to the Halimedineae because utricles. Because of the presence of utricles and the somewhat of shared cytological characters such as heteroplasty. A spongy aspect of the plants, Pseudocodium superficially recent molecular phylogeny on the basis of rbcL gene resembles a Codium. It should be pointed out that certain sequences supports the latter view, although uncertainty Codium species may superficially resemble Pseudocodium to remains regarding its precise position within the suborder the extreme, as is the case with Codium reversum K raft Halimedineae (Lani & Zechman 2006). Under the maxi­ (2007), a delicate species recently described from the Great mum parsimony (MP) criterion Pseudocodium is resolved, Barrier Reef. The complete absence of plugs in the siphons, be it without bootstrap support, as the closest relative of the lack of hairs, lateral cohesion of the utricles and the presence genus Halimeda. Likelihood-based analyses, however, of amyloplasts in Pseudocodium, however, form reliable indicate a sister relationship of Pseudocodium w ith Tyde­ characters that clearly distinguish it from Codium. mania expeditionis Weber-van Bosse. Both the generitype, Pseudocodium devriesii Weber-van * Corresponding author ( [email protected] ). Bosse, and the second (but most likely incorrectly placed) f Present address: Marine Laboratory, University of Guam, species, Pseudocodium australasicum Womersley (1955), are Mangilao GU 96923, USA. known from intertidal and shallow water habitats, but all

225 226 Phycologia, Vol. 47 (2), 2008

Table 1. List of ;-ècL and tufA sequences of Pseudocodium used in this study. New accession numbers are indicated in bold; nonbold accession numbers were previously published by Faye et aí. (2007) or Verbruggen et aí. (2006).

Name Collecting details rht[. tufA P. devriesii Shark Island; Mirbat; Dhofar, Oman (T. Schils, 12.X.2004. DHO2.402) AM909689 AM909694 Reef of Cottesloe, near Perth, Western Australia (D. Mead-Hunter, AM909690 AM909695 19.iv.2001, MURU JH2031) Jesser Point, Sodwana Bay, South Africa (O. De Clerck et cd., 7.xi.2003, AM909691 AM909696 KZN-b 2274) P. floridanum Gulf of Mexico (S. Fredericq, l.vi.2004, NSF.I23) AM909692 AM909697 P. natalense Gotham, Sodwana Bay, South Africa (O. De Clerck et ed., 5.xi.2003, AM909693 AM049969 KZN-b 2241) P. okinawense Nakagusuku Bay, Okinawa, Japan (M. Uchimura, 14.V.2006, Culture C677) AB301930 AM909698 subsequent additions to Pseudocodium have been restricted absence of wet-preserved material from resoaked herbarium to subtidal habitats, some of them growing at the lower specimens. Drawings were made with a camera lucida and boundary of the photic zone. Consequently, the diversity of photographs were taken with an Olympus DP50 digital the genus was largely underappreciated until the introduc­ camera mounted on a Leitz Diaplan compound microscope tion of scuba diving and submersibles became widespread or Leica Wild M10 stereomicroscope. Measurements of tools to study algal diversity in tropical regions. The only utricles are presented as the 95% range of our observations known intertidal species of the genus, P. devriesii, was with absolute minimum and maximum values given described by Weber-van Bosse in 1896 from the Kwazulu- between brackets. Natal coast, South Africa. Some 60 years later, P. New sequences were generated from samples desiccated australasicum Womersley (1955, 1984) was added on the in the field in silica gel or preserved in ethanol (Table 1). basis of collections in the shallow subtidal area from Point DNA was extracted, polymerase chain reaction (PCR)- Sinclair, South Australia. It was not until 1972 that an amplified and sequenced as described by Verbruggen et cd. additional species, Pseudocodium floridanum D aw es & (2005). Amplification of the chloroplast-encoded rbcL and Mathieson, was described on the basis of in situ collections. tufA. genes was based on primers listed in Hanyuda et cd. From that point onward and coinciding with a progres­ (2000) and Farna et cd. (2002) respectively. Generated sively increasing use of scuba diving to study algae in sequences were edited and assembled with M EGA version 4 tropical regions, Pseudocodium has been reported from (T am ura et cd. 2007). Because of missing data at the 5' and several localities in the Indo-Pacifíc (Garrigue & Tsuda 3' termini of the rbcC sequences, a 1335 base pair (bp) 1988; Verheij & Prud’homme van Reine 1993; Wynne 1999; fragment was retained for analysis excluding the first 27 Coppejans et al. 2001, 2005; Lobban & N’Yeurt 2006). and last 96 bp, as compared with the rbcC sequence of However, even though most of the reports have assigned Chlorella vulgaris (Wakasugi et al. 1997). Pseudocodium their specimens to the Caribbean P. floridanum, it is clear rbcL, sequences lacked introns. A 905 bp fragment was that overall habit and utricle morphology are widely analyzed of the tu fA gene. The alignment was exported to divergent between these presumed P. floridanum specimens. PAUP 4.0bl0 (Swofford 2002) for MP and maximum Recently, Faye et cd. (2007) collected a Pseudocodium from likelihood analyses (ML), and MrBayes 3.1.2 (Ronquist & southern Japan, which was described as a new species, Huelsenbeck 2003) for Bayesian inference (Bí). Sequences Pseudocodium okinawense Faye, Uchimura & Shimada. o f Caulerpa racemosa (Forsskâl) J. Agardh, H alimeda Next to morphological differences, rbcC sequences un­ micronesica Y am ada, Halimeda opuntia (Linnaeus) La­ equivocally distinguished P. okinawense from P. floridanum. mouroux and T. expeditionis Weber-van Bosse (accession From this study, the need to thoroughly reinvestigate the numbers AY942161, AB038486, AB038489, AM049971, taxonomy of all recent collections on the basis of AJ512467, AM049964 respectively) were selected as out­ meticulous comparative morphological analyses as well as group taxa. In the MP analyses all characters and character sequence data became apparent. changes were weighted equally. Heuristic searches, consist­ This study examines the diversity of Pseudocodium, ing of 1000 replicates of random sequence addition, were incorporating all described species. Analyses include all performed with tree bisection reconnection (TBR), saving but one of the recently reported Indo-Pacific populations, all minimal trees during branch swapping (MULTREES as well as several additional populations that have not been option activated). MP bootstrap analyses consisted of 1000 hitherto documented. DNA sequences of rhcL and tufA. are replications of full heuristic searches. The model of presented for all species except P. australasicum, an d the nucleotide substitution for ML was selected with Modeltest resulting phylogenies are discussed in an evolutionary and 3.7 (Posada & Crandall 1998) according to the Akaike biogeographical context. information criterion (Posada & Buckley 2004). ML analyses consisted of heuristic searches with 500 random sequence addition replicates and TBR with the option MATERIAL AND METHODS MULTREES in effect. Bootstrap analyses consisted of 500 replications of heuristic searches with the number of Morphological observations were made on specimens rearrangements limited to 10,000 (or 3600 seconds) for preserved in a 5% formalin-seawater solution or in the each replicate, using a neighbor-joining tree as starting tree. D e Clerck et al.: Systematics of Pseudocodium 227

Figs 1-9. Morphology of Pseudocodium natalense. Figs 1-3. Habit of the wet-preserved holotype (KZN-b 2281). Figs 4, 5. Morphology of the truncate and depressed apices. Fig. 6. Surface view of an axis with underlying pigmented medullary siphons. Fig. 7. Surface view of the polygonal and laterally adherent utricles. Fig. 8. Dissected medullary siphons with lateral, broadly clavate reproductive structures (arrows) developing near the base of utricle stipes (arrow heads); note the clear segregation of cytoplasm in the medullary siphons from the densely filled reproductive structure in the stipe of the reproductive structure. Fig. 9. Detail of a medullary siphon showing chloroplasts as well as amyloplasts (darkly staining with lugoi).

Bayesian analyses were performed using a GTR +1 + 1 RESULTS model. The data set was divided in two partitions, corresponding to the first plus second and the third codon Morphological observations positions with all model parameters uncoupled between the Pseudocodium natalense De Clerck, Coppejans & partitions. Posterior probabilities were estimated using a Verbruggen,sp. nov. Metropolis-coupled Markov chain Monte Carlo approach with sampling according to the Metropolis-Hastings Figs 1-9, 10, 14, 20 algorithm. The analysis used four chains, one cold and three incrementally heated. Each run consisted of 2 X IO6 Plantae erectae, virides, e basi filamentosa orientes, at 2 cm generations and was sampled every 100th generation. Burn- alta; laminis complanatis, nonconstricta, 1.0 mm lata et 300- in values were set at 500,000 generations. 350 |T/h crassa. Ramificado dichotoma vel irregularis. Apices 228 Phycologia, Vol. 47 (2), 2008

truncatus et depressus. Fila medullaria 20-60 p/H in diametro, apical pit (Fig. 11). Structure of longitudinally arranged irregulariter constricta et pigmentosa. Utriculius corticales interwoven medullary siphons, 30-50 pm wide, giving rise pius minusve isodiametricae, 70 — 100 X 100 — 125 p/H in to a single layer of peripheral utricles; utricles polygonal diametro, applanatis, cohaerentes, pigmentosa. in surface view (Fig. 15), distinctively clavate in transverse section, (50-) 64—76 (-94) pm in diameter and (153-) 161-186 Plants erect, green in color, arising from a filamentous base, (-200) pm high, with a flat apex, closely laterally adhering up to 2 cm high, consisting of complánate, unconstricted axes, along the entire length of their faces (Fig. 21); utricle stipe 1 mm wide and 300-350 pm thick. Branching dichotomous to not eccentric, often with a distinctive swelling. Chloroplasts somewhat irregular. Apices truncate and depressed. Medullary and amyloplasts present in the utricles and siphons. filaments or siphons 20-60 pm in diameter, irregularly con­ Reproductive structures immersed in the thallus, produced stricted and pigmented. Cortical utricles more or less isodiamet- laterally from medullary siphons close to the base of the ric, 70—100 X 100—125 pm in diameter, flat-topped, coherent utricle stipe, ovoid to irregularly shaped, 80-130 pm in and pigmented. diameter (Fig. 22).

H O LO TY PE: KZN-b 2281 (GENT), collected by H. Verbrug­ TYPE: Isipingo Beach, near Durban, Kwazulu-Natal, South gen and T. Schils at —33 m on the slope of Wright Canyon, Africa (Weber van Bosse, 1894—1895, L 936.73-447). Sodwana Bay, northern Kwazulu-Natal, South Africa on 7 November 2003. See Coppejans et al. (2005, as P. floridanum) DISTRIBUTION AND HABITAT: Pseudocodium devriesii is a for an in situ photograph of the type specimen. common to abundant species in South Africa, southern Mozambique and the warm temperate part of Madagascar. It description : Thallus upright, to 2 cm high, anchored in grows in the lower part of the intertidal area, often in tidal sandy substrate by basal, branched, multicellular rhizoids pools, as well as in the shallow subtidal area. Pseudocodium (Figs 1-3), bright green in colour; axes compressed, 1 mm devriesii is less common in Oman and Western Australia, wide and 300-350 pm thick, two to four times dichoto- where only a few populations are known that are moreover mously branched, hardly tapering toward the apices, lacking confined to the subtidal area (8-15 m depth). constrictions; apices truncate with a depressed apical pit (Figs 4, 5, 10). Thalli composed of longitudinally arranged, s p e c i m e n s e x a m i n e d : South Africa(selected): Cape Mor­ interwoven, irregularly swollen medullary siphons, 20- gan (O. De Clerck, 25.x.1999, K ZN 1293); Port Edward (F. 60 pm wide, giving rise to a single layer of peripheral Leliaert, 24.iii. 1997, FL 339); Isipingo, D urban (E. Coppe­ utricles (Fig. 6); utricles polygonal in surface view (Figs 5, 7, jans, 21.xi.1995, HEC 10948); Mission Rocks ( O. De Clerck 14), with a flat apex (Fig. 20), approximately isodiametric in & F. Leliaert, 13.vi.2003, KZN 2257); Jesser Point, Sodwana transverse section, (71 — ) 80-88 (-101) pm in diameter and Bay (O. De Clerck, T. Schils, H. Verbruggen & E. (104) 112-123 (126) pm high, laterally adhering except at the Demeulenaere, 7.xi.2003, KZN-b 2247); Kosi Bay ( E. corners; utricle stipe not eccentric. Chloroplasts and Coppejans et al, 16.viii.l999, KZN 774). Mozambique, amyloplasts at least in living plants present in the utricles Cabo Inhaca (E. Coppejans, 18.xii.2000, HEC 14263). and siphons (Fig. 9). Reproductive structures immersed in Madagascar, Fort Dauphin (E. Coppejans, D. Douterlugne, the thallus, produced laterally from medullary siphons close I. Razanakoto, 30.viii.2002, HEC 15195). Oman: H oon’s to the base of the utricle stipe, variable in morphology, Bay, Mirbat; Dhofar (71 Schils, 9.X.2004, DH02.274); ranging from spherical on a gradually broadening stipe to Shark Island, Mirbat, Dhofar (71 Schils, 12.X.2004. irregular, broadly clavate structures, 80-90 pm wide and 120- DHO2.402 and DHO2.403). Australia:Western Australia, 185 pm long, lacking a basal cross wah separating it from the Reef off Cottesloe, near Perth, from 8-9 m depth (D. Mecid- rest of the thallus, opening distally by an undifferentiated pore Hunter, 19.iv.2001, PERTH 07259697); Rous Head, near (Fig. 8). Fremantle, from 8 m depth (71 Huisman, 25.iii.2007, PERTH 07561296). o t h e r s p e c i m e n s EXAMINED: South Africa: Gotham , Sodwana Bay, — 3 0 m (O. De Clerck, T. Schils, H. n o t e s : Preliminary culture studies of Western Australian Verbruggen & E. Demeulenaere, 5.XÍ.2003, KZN-b 2 2 4 1 ) ; plants (Huisman, unpubl. obs.) indicate that basal siphons Tombstone, 5-Mile Reef, Sodwana Bay, — 3 0 m (O. De Clerck, T. Schils, H. Verbruggen & E. Demeulenaere, can extend horizontally for some distance and then produce 6.xi.2 0 0 3 , KZN-b 2 2 5 0 ) . new upright thalli. Thus the clusters of plants that are often observed may be clonal. DISTRIBUTION AND HABITAT: Pseudocodium natalense is thus far only known from Sodwana Bay, South Africa, where it is Pseudocodium floridanum Dawes & Mathieson, 1972: 273 confined to deep water (below —30 m depth). The species is rather common, growing on coarse sand and coral debris. Figs 13, 17, 18

description : Thallus upright, to 10 cm high, anchored in Pseudocodium devriesii Weber van Bosse, sandy substratum by a well-developed tuft of rhizoids, bright 1896: 209-212, pi. I. green in colour; axes cylindrical, to 5 mm wide near the base, Figs 11, 15, 21, 22 gradually attenuating toward the apices, dichotomously to somewhat irregularly branched, unconstricted; apices round­ description : Thallus upright, to 5-8 (-14) cm high, attached ed. Thalli composed of longitudinally arranged interwoven to rocks by a feltlike rhizoidal holdfast, bright green in colour and irregularly swollen medullary siphons, 50-110 |im wide, (drying darker); axes compressed or subterete and becoming giving rise to a single layer of peripheral utricles; utricles compressed in the lower portions, 0.9-2.5 mm wide and 0.5- polygonal in surface view, with a broadly rounded apex 1.0 mm thick, dichotomously branched up to seven times, (Figs 13, 17), distinctively broader than high in transverse tapering slightly or not at all toward the apices, often with section, (143-) 248-310 (-365) pm in diameter and (177-) undulate margins; irregular shallow constrictions of the axes 187-215 (-225) pm high, adhering laterally to neighbouring uncommon to prominent; apices truncate with a depressed utricles except at the corners; utricle stipe eccentric (Fig. 18). D e Clerck et al.: Systematics of Pseudocodium 229

10-13

14-16 17 Figs 10-17. Comparative morphology of apices and utricles in surface view. Figs 10, 14 = P. natalense ; Figs 11, 15 = P. devriesii'. Figs 12, 16 = P. okinawense'. Figs 13, 17 = P. floridanum (after Dawes & Mathieson 1972). Scale bars = 250 pm.

Chloroplasts and amyloplasts absent from utricles and ellipsoidal in shape, 80-120 pm in diameter, lacking a basal confined to the medullary siphons. Reproductive structures cross wall separating them from the rest of the thallus (after immersed in the thallus, produced laterally from medullary Dawes & Mathieson 1972). siphons close to the base of the utricle stipe, spherical to TYPE: 38 nautical miles west of Egmont Key, mouth of Tampa Bay, Florida (C.J. Dawes, l.ix.1967, 6280A, not examined).

distribution AND HABITAT: The species is known from several localities in the Gulf of Mexico, the Atlantic coast of Florida and the Bahamas, where it may form extensive perennial stands on sandy bottoms with up to 45 individuals per square meter at depths varying from 24 to 60 m (Dawes & Mathieson 1972; Flanisak & Blair 1988; N orris & Olsen 1991).

s p e c i m e n s e x a m i n e d : Palm Beach, Florida, USA: (leg. M. & D. Littlei-, 14.viii.2006, US DML58944); dredged at 24°36.48'N, 83°24.77'W in the Gulf of Mexico, -60 m (S. Frederici] et a l, l.vi.2004, NSF-I-23); dredged at 21°33.97'N, 91°04.75'W deep in Bahia de Campeche, Gulf of Mexico, — 32 m (S. Frederici] et al., 12.vi.2005, NSF-II-57-7); dredged at 28°55.12'N, 85°09.62'W off the coast of Florida in the G ulf of Mexico, —54 m (S. Fredericq et al, 7.iii.2006, NSF-III-7/3/06/-9-2).

Pseudocodium okinawense Faye, Uchimura & Shimada, 2008:105

Figs 18-21. Comparative morphology of utricles in lateral view. Figs 12, 16, 19 Fig. 18 = P. floridanum (after Dawes & Mathieson 1972 ); Fig. 19 = P. okinawense'. Fig. 20 — P. natalense'. Fig. 21 = P. devriesii. Scale description : Thallus upright, to 1-2.5 cm high, anchored bars =100 pm. in sandy substratum by a basal tuft of rhizoids, olive- to 230 Phycologia, Vol. 47 (2), 2008

dark green in colour; axes terete, simple to 3(—4) times dichotomously branched, 0.5-1 mm in diameter, hardly tapering toward the apices, unconstricted; apices rounded (Fig. 12). Thalli composed of longitudinally arranged interwoven medullary siphons, 20-60 pm wide, giving rise to a single layer of peripheral utricles; utricles polygonal in surface view, with a rounded apex (Fig. 16), approximately isodiametric in transverse section, (70-) 128-147 (-187) pm in diameter and (113—) 132-147 (-172) pm high (Fig. 19), adhering laterally to neighbouring utricles except at the corners; utricle stipe not eccentric. Chloroplasts and amyloplasts predominantly present in the siphons. Repro­ ductive structures not observed.

TYPE: Nakagusuku Bay, Awase, Okinawa, Japan (M. Uchimura, 14.V.2006. SAP 1 0 2 8 1 7 ) .

DISTRIBUTION AND HABITAT: Pseudocodium okinawense is known from Indonesia, Papua New Guinea and the Ryukyu Islands in southern Japan. Most collections were made in Fig. 22. Reproductive structures of P. devriesii developing near the relatively deep habitats, —30 to —40 m, but Japanese base of the utricle stipes. Scale bar = 100 pm. specimens appear to grow in shallower parts of the coastline, between —8 and —12 m.

s p e c i m e n s e x a m i n e d : Indonesia:Samalona Island, Sulawesi Pseudocodium australasicum Womersley, (E. Verhei], 3.X.1989, L 992.261-133); Samalona Island, 1995: 390, Figs 5, 6 Sulawesi (E. Verheij, l.ix.1989, L 992.261-149). Japan: Awase, Okinawa (M. Uchimura, l.v.2007, H.0923); Awase, Figs 23-26 Okinawa (M. Uchimura, 22.L2007, H.0924); Awase, Oki­ nawa (M. Uchimura, 6.vi.2007, H.0925). Papua New Guinea: Thalli erect, up to 6 cm high, with subdichotomous to Laing Island (E. Coppejans, 22.vi.1980, HEC 4392); Hansa irregularly branched complánate axes, 1-2 mm broad, dark Bay (E. Coppejans, 13.viii.1980, HEC 4648). green in colour (Figs 23, 24). Thalli composed of longitu­ dinally arranged interwoven medullary siphons, 13-30 pm in n o t e : Specimens from Indonesia and Papua New Guinea diameter, beset with peripheral appendages that consolidate were previously identified as P. floridanum by Verheij & to form a continuous cortex; appendages more or less Prud’homme van Reine (1993) and Coppejans et al. (2001), perpendicular to the long axes of the thallus, 40-90 pm long, not constricted at the base, ending in a cluster of digitations, respectively. These specimens correspond very well to the 7-12 pm wide at the tips (Figs 25, 26). Apices rounded, not Japanese species, be it that they are slightly larger and truncate. Chloroplasts and amyloplasts present. Reproduc­ generally more luxuriantly branched. This can possibly be tive structures not observed, (partially after Womersley attributed to the fact that the Ryukyu Islands (also referred 1955, 1987). to as Nansei Islands or Nansei Shoto) most likely represent the most northern distribution limit of this tropical species. TYPE: Point Sinclair, South Australia (H.B.S. Womersley, Specimens from Indonesia are characterized by utricles that 25.i.1951. AD-A13618). are marginally smaller compared with the specimens from DISTRIBUTION AND HABITAT: Pseudocodium australasicum is Papua New Guinea, measuring on average 80-120 pm in known only from sporadic collections spanning the southern diameter. Those values, however, fall completely within the Australian coastline from Hopetoun, Western Australia to range of 50-200 pm indicated for P. okinawense by Faye et Point Sinclair. Collections were made either from the drift or al. (2007), 50-200 pm. Given the fact that the Indonesian from the upper sublittoral. specimens correspond in ali other aspects to the Japanese s p e c i m e n s e x a m i n e d : Australia:Hopetoun, Western Aus­ and Papua New Guinean specimens, we attribute these tralia (G.T. Kraft & R. Ricker, 16.viii.1979, ADU-A50717; specimens to P. okinawense. The specimen reported as duplicate wet preserved material provided by G.T. Kraft). Pseudocodium sp. from Guam (Lobban & N’Yeurt 2006) was not available for study. On the basis of the following Molecular phylogenetics observations from Lobban (2007), however, the sample perfectly corresponds to the description of P. okinawense: The molecular data set consisted of rbcV (1335 bp) and small habit (< 2 cm), narrow (< 1 mm) and terete thallus tufA. (905 bp) sequences of 6 Pseudocodium isolates and 3 morphology, basal tuft of rhizoids (‘cushion-like base’), and outgroup taxa ( Caulerpa, Halimeda and Tydemania). O f a loosely adhering subspherical utricles (see also Fig. 19). If total of 2240 characters, 343 were parsimony informative the sample indeed represents P. okinawense, this would (187 bp in rbcL and 156 bp in tufA ). The phylogenetic tree increase the species’ depth range considerably to at least inferred from a combined analysis of rbcL and tu fA 75 m. Moreover, Garrigue & Tsuda (1988) reported a sequences is shown in Fig. 27. Support values of the genus Pseudocodium sp. from New Caledonia in the South Pacific. and the intrageneric relationships are high. Trees of both These potential records indicate that the recently described genes analyzed separately have identical topologies (results P. okinawense might possess the largest contiguous not shown ). Only under MP two trees of minimal length are distribution area. inferred that differ in the placement of P. okinawense, either D e Clerck et al.: Systematics of Pseudocodium 231

50 pm

Figs 23—26. Morphology of Pseudocodium australasicum. Fig. 23. Habit of a wet-preserved specimens from Hopetoun, Western Australia (duplicate of ADU-A50717). Fig. 24. Detail of the apices. Figs 25, 26. Dissected medullary siphons with lateral appendages ending in clusters of digitations. as the sister taxon of P. floridanum or as the closest relative DISCUSSION o f the P. devriesii - natalense clade. This result was obtained from the rbcL as well as tufA. data set. A combined analysis The taxonomic position of Pseudocodium was only recently o f rbcL and tu fA sequences under the MP criterion, confirmed on the basis of rbcL gene sequences. The genus however, results in a single most parsimonious tree with was solidly placed in the Halimedineae (Lani & Zechman P. okinawense as the sister species of P. floridanum. The 2006). Besides the molecular results there are several newly described species, P. natalense, is invariably resolved additional morphological characters that support this as the sister species of P. devriesii. The uncorrected genetic placement: the polygonal aspect of the utricles when distance between these two species is rather low; being 2%. observed in surface view, the lateral cohesion of the utricles This is markedly lower than the distances of 12-13% and heteroplastidy and the absence siphon plugs. The separating the other species. Geographical isolates of P. vegetative morphology of P. australasicum is clearly at odds devriesii form a monophyletic lineage, with the Omani with the other species of the genus. Most importantly, P. specimens slightly divergent from the South African and australasicum doesn’t possess a cortex composed of Western Australian specimens, which share identical inflated, globose utricles. Rather, peripheral siphons form sequences. The Omani specimen differs at 6 sites (approx. laterals that branch repeatedly and form clusters of tight 0.3%), 2 in the rbcL gene and 4 in the tu fA sequence. digitations that consolidate in a cohesive cortex. These characters are reminiscent of certain Udotea species

— P. natalense (South Africa) characterized by fully corticated blades, e.g. Udotea 1O3ÍT0OÍ1M flabellum (Ellis & Solanderi Howe and U. norisii D.S. r P. devriesii (Om an) P. dewiesii (South Africa) Littler & M.M. Litller (Gepp & Gepp 1911; Littler & Littler iooï93na[> Pseudocodium ei ' Tenml P d e b e s ii (Australia) 1990; Kraft 2007). Contrary to Udotea, the thallus of P.

P. okinawense (Japan) australasicum is not calcified. At this stage it seems justified P. floridanum (Florida. USA) to exclude P. australasicum from Pseudocodium. W e do Tydemania refrain, however, from formally transferring the species to

■ Halimeda outgroups the Udoteaceae for the following reasons: (1) the lack of Caulerpa sequence data that would offer additional support for a formal transfer; (2) generic concepts of the Udoteaceae are in itself in need of critical revision, with several genera being Fig. 27. Maximum likelihood phylogeny of the genus Pseudocodium poly- or paraphyletic (Kooistra 2002). on the basis of combined rbcC and tufA sequences, rooted with Caulerpa and Halimeda and Tydemania. Node support values are Holocarpy, the type of reproduction whereby the entire given at each ramification of the tree (MP/ML/BI). The log- thallus is transformed into spores or gametes, was long likelihood of the tree is 7852.65. Base frequencies are A = 0.311, C believed to be an additional synapomorphy of the suborder. = 0.149 G = 0.220, T = 0.320. The substitution rates are AC = Observations on Caulerpella, the sister genus of Caulerpa, by 1.715, AG = 2.256, AT = 2.490, CG = 0.774, CT = 5.676, GT = 1. The proportion of invariable sites in the alignment is 0.3713 and Prud’homme van Reine & Lokhorst (1992), made it clear the shape parameter of the gamma distribution among site rate that holocarpic reproduction is not a universal feature of the heterogeneity is 0.656. Halimedineae. In Pseudocodium putative reproductive struc- 232 Phycologia, Vol. 47 (2), 2008

Table 2.Diagnostic characteristics of Pseudocodium (excluding P. australasicum).

P. devriesii P. floridanum P. natalense P. okinawense Height ■si 10 cm si 10 cm ss 2 cm ss 2 cm Axes Morphology subterete to compressed terete compressed terete Diameter to 2.5 mm to 5 mm to 1 mm to 1 mm Constrictions present absent absent absent Apex depressed rounded depressed rounded Utricles Coherence laterally adhering closely laterally adhering adhering laterally relatively loosely except at the corners along the entire length except at the corners adhering Apex flat rounded flat rounded Diameter in surface view (50-) 64-76 (-94) pm (143 ) 248-310 (-365) pm (71-) 80-88 (-101) pm (70-1 128-147 (-187) pm Shape and height in rectilinear (15 3 ) cushion-shaped (177-1 isodiametric (104) spherical (113-1 132-147 transverse section 161-186 (-200) pm 187-215 (-225) pm 112-123 (126) pm (-172 ) pm Plastids in utricles (in situ) present absent (?) present absent Depth range intertidal to shallow >24m >30m 8M0 m (-75 m) subtidal Distribution subtropical Indian Caribbean Sea and South Africa western Pacific Ocean tropical Atlantic Ocean tures were so far only reported for P. floridanum (Dawes & flat-topped. Those of P. devriesii are markedly longer than Mathieson 1972). Observations on P. devriesii an d P. wide in transverse section, whereas utricles of P. natalense natalense reveal very similar structures, formed laterally on are typically isodiametric. The utricle apices of P. medullary siphons. Interestingly and confirming the original floridanum and P. okinawense are rounded. The shape of observations of Dawes & Mathieson (1972), no basal cross the apex of the axes forms another character uniting both wall is formed at the base of these structures. Observation of couples of species. Pseudocodium floridanum and P. structures from which the content was released by means of okinawense have both a rounded apex, whereas the apices an apical pore reveal the presence of a translucent plug o f P. devriesii and P. natalense are distinctively truncate entirely filling its stipe. W ithin the Bryopsidales, gametangial with an apical depression. The latter two species are also plugs are important diagnostic features, e.g. to discern the characterized by compressed axes. They are terete in P. genus Pseudobryopsis from the reinstated genus Tricho­ floridanum and P. okinawense. The position of the solen (Henne & Schnetter 1999). These plugs prevent the chloroplasts in the thallus might present an additional cytoplasm from leaking into the environment. Under the diagnostic character separating both clades. Dawes & assumption that these structures represent gametangia, this Mathieson (1972) and Faye et al. (2007) state explicitly indicates that Pseudocodium is not holocarpic, contrary to that medullary siphons are densely filled with chloroplasts, most Halimedineae. Other circumstantial evidence is also whereas utricles are nearly devoid of plastids in both P. against holocarpy in Pseudocodium. For example, unlike in floridanum an d P. okinawense. In situ pictures of P. devriesii Halimeda, Udotea and Chlorodesmis, the entire cytoplasm o f and P. natalense clearly show that chloroplasts are present the plant does not retreat in the gametangia. Furthermore, in the utricles as well (Coppejans et cd. 2005; J.M. Huisman, holocarpic reproduction would result in ghost thalli, unpublished observations; see also Guiry & Guiry 2007). leftovers of fertile thalli entirely devoid of cytoplasm. It is However, in formalin-preserved specimens of P. devriesii likely that such ghost thalli would have been observed at and P. natalense plastids are primarily restricted to the least for P. devriesii, which is one of the more common medullary filaments. Since chloroplasts are known to intertidal species along the South African coastline. It should migrate actively in siphonous algae (Drew & Abei 1990; be pointed out, however, that due to a lack of observations Kooistra 2002), the absence of chloroplasts from utricles in on cultured plants these structures were never examined in preserved specimens could therefore represent an artifact. sufficient detail to fully ascertain that they are gametangia, Nevertheless, in situ observations on P. floridanum w ould sporangia or even some kind of propagule aiding in be of value ascertaining the reliability of this character. vegetative multiplication. Re-examination of P. floridanum records from the Indo- Morphological re-examination of most Pseudocodium Pacific Ocean results in a better understanding of the specimens that have been reported in the last two decades, biogeography of the individual species (Fig. 28). Reports of combined with gene sequences of all known species (except P. floridanum from the western Pacific Ocean (Verheij & P. australasicum), result in a much clearer picture of the Prud’homme van Reine 1993; Coppejans et cd. 2001) diversity of the genus. A combination of characters relating represent misidentifications and the specimens are repre­ to general habit, morphology of the axes, apices and utricles sentative of P. okinawense, a species recently described from distinguishes all four accepted species easily (Table 2). In an the Ryukyu Islands. Likewise, the report of P. floridanum evolutionary context one can discern two lineages that are from South Africa is also incorrect. These specimens belong each supported by a whole suite of morphological to a new species, P. natalense, described in this paper. characters. Utricles in P. devriesii and P. natalense are G enuine P. floridanum is most likely restricted to the D e Clerck et al.: Systematics of Pseudocodium 233

;; P. floridanum III P. de-vriesii = P. natalense :: P. okinawense Fig. 28. Global distribution of Pseudocodium species. tropical western Atlantic and Caribbean region. Pseudoco­ and P. natalense on the other hand diverge by only 2%, dium okinawense probably has a wider distribution than the indicating a much more recent origin of these two species. current records indicate. Its diminutive size and preference This study expands the distribution of P. devriesii for relatively deep subtidal habitats make it likely that the considerably. The species was previously known from the species is overlooked in the field. More extensive sampling subtropical southwestern Indian Ocean (South Africa, the may result in a tropical western Pacific distribution pattern, southernmost point of Mozambique and Madagascar; Silva which may or may not extend in the Indian Ocean. et cd. 1996) and Oman (Wynne 1999). Here the distribution Despite the fact that only four extant species of Pseudo­ range o f P. devriesii is extended to Western Australia, which codium are known, their biogeographic distribution pattern makes it an interesting species to study disjunct distribu­ is an inviting one and leads us to speculate about the time tions in the Indian Ocean. Biogeographic links between and place of their diversification. A first split in the distant subtropical regions in the Indian Ocean have been phylogenetic tree separating P. floridanum — okinawense repeatedly suggested in the literature (e.g. Borgesen 1934; from P. devriesii - natalense coincides with an ecological Silva 1962; Norris & Aken 1985; Hommersand 1986; differentiation leading to a tropical and a more temperate F arrell et cd. 1994; Wynne 2000; Schils & Coppejans lineage. The sister relationship between P. floridanum and P. 2003). Unfortunately, the widespread misapplication of okinawense could be explained as a vicariant event separat­ algal names, due to an inadequate taxonomic framework ing tropical Atlantic and Indo-Pacific lineages. We hypoth­ and high levels of pseudocryptic diversity among marine esize that this diversification coincided with the closure of macroalgae, makes it perilous to deduce biogeographical the tropical Tethys Sea. A similar scenario whereby affinities on the basis of species lists. Consequently, many ecological differentiation predates geography-based spécia­ of these presumed biogeographic links do not hold when tion is also represented by the calcified green algal genus sequence data are used to test these hypotheses. Verbruggen H alimeda (Kooistra et cd. 2002; Verbruggen et cd. 2005). For et cd. (2007) demonstrated convincingly that in the genus the latter genus, Verbruggen et cd. (2005) associated this Codium many links are artificial and that morphological vicariant event with the final closure of the Tethys Sea in the similarities can be most likely attributed to convergent Miocene. Contrary to Pseudocodium, the calcified segments evolution. However, DNA sequences did reveal a number o f H alimeda left a reasonably detailed footprint in the of cases that would support a clear evolutionary link paleorecord, which supports its differentiation in the Tethys between subtropical Indian Ocean floras. Specimens of Sea. As for Pseudocodium, lacking fossils, we can only guess Codium duthieae as well as Halimeda cuneata from South whether it was widely distributed in the Tethys Ocean. East Africa, Oman and Western Australia do form Uncorrected p-distances between the P. floridanum - monophyletic lineages, reflecting a common origin of these okinawense clade and the clade uniting the two subtropical geographically separated populations (Verbruggen et cd. 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