J. Phycol. 37, 881–899 (2001) SYSTEMATICS OF THE DELESSERIACEAE (CERAMIALES, RHODOPHYTA) BASED ON LARGE SUBUNIT rDNA AND rbcL SEQUENCES, INCLUDING THE PHYCODRYOIDEAE, SUBFAM. NOV.1 Showe-Mei Lin,2 Suzanne Fredericq3 Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana 70504-2451 and Max H. Hommersand Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280 The present classification of the Delesseriaceae research promotes the correlation of molecular and retains the essential features of Kylin’s system, which morphological phylogenies. recognizes two subfamilies Delesserioideae and Ni- Key index words: Ceramiales; Delesseriaceae; LSU tophylloideae and a series of “groups” or tribes. In rDNA; rbcL; Phycodryoideae subfam. nov.; Deles- this study we test the Kylin system based on phyloge- serioideae; Nitophylloideae; Rhodophyta; systemat- netic parsimony and distance analyses inferred from ics; phylogeny two molecular data sets and morphological evidence. A set of 72 delesseriacean and 7 additional taxa in Abbreviations: LSU, large subunit the order Ceramiales was sequenced in the large sub- unit rDNA and rbcL analyses. Three large clades were identified in both the separate and combined The Delesseriaceae is a large family of nearly 100 data sets, one of which corresponds to the Deles- genera found in intertidal and subtidal environments serioideae, one to a narrowly circumscribed Nitophyl- around the world. Kylin (1924) originally recognized loideae, and one to the Phycodryoideae, subfam. nov., 11 groups in the Delesseriaceae that he assigned to two comprising the remainder of the Nitophylloideae subfamilies: Delesserioideae (as Delesserieae) and Ni- sensu Kylin. Two additional trees inferred from rbcL se- tophylloideae (as Nitophylleae) based on the location quences are included to provide broader coverage of of the procarps (whether restricted to primary cell rows relationships among some Delesserioideae and Phyco- or scattered over the thallus surface), the presence or dryoideae. Belonging to the Delesserioideae are the absence of midribs with rhizoidal filaments, and the Caloglosseae with Caloglossa; an expanded Hem- presence or absence of intercalary cell divisions in the ineureae that includes Hemineura, Patulophycus, Mar- primary cell rows. ionella, Laingia, Botryocarpa, and Pseudophycodrys; the The Delesserioideae were further divided by Kylin Delesserieae with Delesseria and Membranoptera; the into groups as follows: (1) intercalary cell divisions ab- Apoglosseae with Apoglossum and a group of south- sent in all cell rows and all third-order cell rows reach- ern hemisphere species presently placed in Delesseria ing the blade margin (Hypoglossum group), (2) in- that belong in Paraglossum; the Hypoglosseae with Hy- tercalary cell divisions absent in all cell rows but not poglossum, Branchioglossum, Zellera, and Bartoniella; all third-order cell rows reaching the blade margin and the Grinnellieae with Grinnellia. The revised Ni- (Membranoptera group), (3) intercalary cell divisions tophylloideae contains the Nitophylleae with Nitophyl- present in second- and higher order cell rows with the lum, Valeriemaya, Polyneuropsis, and Calonitophyllum procarp consisting of one carpogonial branch (Deles- and the Martensieae with Opephyllum and Martensia. seria group), (4) as above, but the procarp consisting A new subfamily, Phycodryoideae, is proposed to in- of two carpogonial branches (Hemineura group), clude the Phycodryeae with Phycodrys, Polyneura, Nien- and (5) intercalary cell divisions present in second- burgia, Cladodonta, Heterodoxia, and Womersleya; the and higher order cell rows with the procarps pro- Cryptopleureae with Cryptopleura, Hymenena, Acroso- duced on minute bladelets immersed in the surface of rium, and Botryoglossum; the Myriogrammeae with Myrio- the parent blade (Grinnellia group). gramme and Haraldiophyllum; and the Schizoserideae Groups in the Nitophylloideae were characterized with Schizoseris, Neuroglossum, Drachiella, Abroteia, and as follows: (1) growth by means of a transversely divid- species from South America placed in Platyclinia. This ing apical cell and midribs with downward coursing rhizoids (Pseudophycodrys group), (2) as above, but midribs lacking rhizoids (Phycodrys group), (3) as in Phycodrys, but branching from the midrib (Ruprechti- 1 Received 24 January 2001. Accepted 24 June 2001. ella group), (4) growth by marginal meristematic cells, 2 Present address: Research Department, National Museum of Ma- rine Biology and Aquarium, 2 Houwan Rd., Checheng, Pingtung 944, microscopic veins present (Cryptopleura group), (5) Taiwan. growth by marginal meristematic cells, microscopic 3 Author for correspondence: e-mail [email protected]. veins absent (Myriogramme group), and (6) as above, 881 882 SHOWE-MEI LIN ET AL. but the procarp with only one sterile group (Nitophyl- quenced in this study are listed in Table 1, third column. lum group). Vouchers extracted for DNA analysis include all taxa sequenced in this study, which are listed in Table 2, together with their Taxonomic concepts used in the current classifica- GenBank accession numbers. tion of the Delesseriaceae have changed little since DNA samples were prepared using the DNeasy Plant Mini the first proposals by Kylin (1924). Kylin (1956) added Kit (QIAGEN, Valencia, CA) or were submitted to a hexadecyltri- the Sarcomenia group, the Claudea group, the Botry- methylammonium bromide (CTAB)-cesium chloride DNA pro- cedure (Freshwater et al. 1994). The addition of 0.5 ϫ 10Ϫ3 to ocarpa group, the Papenfussia group, and the Mar- Ϫ 1 ϫ 10 3 mg of proteinase K to each extraction when grinding tensia group and renamed the Ruprechtiella group as the samples using the Qiagen Minikit often improved the DNA the Yendonia group based on a suite of vegetative char- yield (Hughey and Hommersand 1999). acters and procarp architecture. To these Wynne (1983) The genes selected to infer the phylogeny of the Delesseri- added four additional groups to the Delesserioideae: aceae are chloroplast- encoded rbcL and the nuclear-encoded LSU rDNA gene. The rbcL primers used in this study are as the Congregatocarpus group with intercalary divisions listed in Freshwater and Rueness (1994) and Hommersand et in primary cell rows and three sympodially branched al. (1994), and the LSU rDNA primers used in this study are groups, the Sympodophyllum group with intercalary listed in Freshwater et al. (1999). The entire rbcL-coding region divisions absent, the Cumathamnion group with inter- was sequenced except the first 84 base pairs, whereas the first calary cell divisions present only in second-order cell 300 base pairs and the last 1200 base pairs of the LSU rDNA- coding region were not sequenced due to lack of informative rows, and the Kurogia group with intercalary cell divi- signal. Additional internal sequencing primers designed specif- sions also present in first-order cell rows. The Yen- ically for the Delesseriaceae include donia group was merged with the Phycodrys group. LSU-F449 (5Ј-CCCGAAAGA TGGTGAACTATG-3Ј) Wynne (1996) published a revised key to genera of LSU-R831 (5Ј-GAATGCCAAGTA GGGCATAGC-3Ј) Delesseriaceae together with a list of the genera, type rbcL-F64 (5Ј-CCATATGCAAAAATGGGATAC-3Ј) species, and recent references and added the Valeri- rbcL-F645 (5Ј-ATGCGTTGG AAAGAAAGATTCT-3Ј). emaya group (Millar and Wynne 1992) to the subfam- For gene amplification, 2 ␮L of the resulting extractions ily Nitophylloideae based on the presence of a persis- were used as templates for a 50-␮L PCR consisting of 10 ␮L 5 M tent apical cell. Three tribes have been proposed or betaine, 6 ␮L 10␮ PCR buffer (Perkin Elmer Corp., Foster City, ␮ ␮ emended since 1996: the Dicroglosseae (Millar and CA), 6 L 25 mM MgCl2 solution, 8 L of 500 mM dNTP stock, 2 ␮L each of the appropriate primers at 10 mM, and 0.3–0.5 ␮L Huisman 1996), the Myriogrammeae (Hommersand Amplitaq DNA Polymerase (PE Applied Biosystems, Foster City, and Fredericq 1997a), and the Schizoserideae (Hom- CA). Amplification conditions for both rbcL and LSU rDNA mersand and Fredericq 1997b) (Table 1). Recently, consisted of 4 min at 96Њ C for denaturation, followed by 35 cy- Wynne (2001) extended formal recognition at the cles of 60 s at 94Њ C, 60 s at either 45Њ C or 42Њ C, and 90 s at 72Њ C, with a final 8-min extension cycle at 72Њ C, and soak cycle at tribal level to all 23 groups and tribes presently identi- 4–12Њ C. The amplification reactions were performed on a PE fied in the Delesseriaceae. GeneAmp PCR system 9700 or 2400 (PE Applied Biosystems). A main goal of this study was to test the Kylin Some PCR amplifications that initially failed were successfully (1956) and Wynne (1996, 2001) systems (Table 1) amplified under one-half volume conditions by doubling Amp- and to evaluate phylogenetic relationships at subfam- litaq DNA Polymerase or by increasing the PCR to 42–45 cycles. For automated gene sequencing, amplification products ilial, tribal, and generic levels based on large subunit were cleaned of excess primer, enzyme, and dNTPs by PEG pre- (LSU) rDNA and rbcL molecular data sets and a reas- cipitation (Hillis et al. 1996). The sequences were determined sessment of the morphological evidence. over both strands using an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems) with the ABI Prism BigDye Terminator Cy- cle Sequencing Ready Reaction Kit (PE Applied Biosystems). materials and methods Reaction mixtures comprised 4 ␮L Terminator