Zosteraceae) in Australia and New Zealand

Home , Phyllospadix, Potamogetonaceae, Zostera, Zosteraceae, Zostera capricorni, Zostera caulescens

Systematics of Seagrasses (Zosteraceae) in Australia and New Zealand

DONALD H. LES,1,4 MICHAEL L. MOODY,1 SURREY W. L. JACOBS,2 and RANDALL J. BAYER3 1Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut 06269-3043; 2Royal Botanic Gardens, Sydney, NSW, 2000, Australia; 3CSIRO, Division of Plant Industry, Australian National Herbarium, GPO Box 1600, Canberra, ACT, 2601, Australia 4Author for correspondence ([email protected])

Communicating Editor: Jeff H. Rettig

ABSTRACT. Previous taxonomic treatments of the family Zosteraceae in Australia/New Zealand have recognized Heter- ozostera tasmanica (monotypic) and four Zostera species all belonging to subgenus Zosterella: Z. capricorni, Z. muelleri, Z. mucronata, Z. novazelandica. Zostera has always been taxonomically problematic in Australia, where researchers have expressed difficulty with species recognition due to vague or inconsistent morphological characters. There also has been a lack of agreement on generic (notably the distinctness of Heterozostera) and subgeneric delimitation. Recent anatomical, developmental, and molecular studies urge a reevaluation of relationships in the family. To clarify the taxonomy of Zosteraceae, we investigated interspecific phylogenetic relationships focusing on Australian species of subgenus Zosterella. We examined material comprising all genera of Zosteraceae (Heterozostera, Nanozostera, Phyllospadix, Zostera), six/seven species of Zostera subgenus Zosterella (including all Australian/New Zealand species), and one of four species of Zostera subgenus Zostera.We conducted phylogenetic analyses of morphological data and DNA sequences from nuclear (ITS) and plastid (trnK intron, rbcL) genomes. Our results indicate two major clades (highly divergent at both morphological and molecular levels) and two subclades (with low morphological and molecular divergence) within Zosteraceae. Little morphological and molecular variation was observed among representatives within the clade of Australian/New Zealand members of subgenus Zosterella, and none provided cladistic support for taxa recognized formerly as separate species. We recommend that Zosteraceae comprise two genera (Phyllospadix, Zostera) with the latter subdivided into three subgenera (Zostera, Zosterella, Heterozostera). Furthermore, Australian/New Zealand representatives of Zostera subgenus Zosterella should be merged within a single species (Z. capricorni) to reflect the inability of morphological or molecular data to effectively delimit additional species in this group.

The marine, monocotyledonous Zosteraceae Du- tera ...’’buthada‘‘different way of branching’’ and mort. are typically circumscribed to comprise three a distinctive rhizome vasculature. Incorporating these genera (Heterozostera, Phyllospadix, Zostera) with ap- features into his key, Hartog distinguished Heterozos- proximately 18 species (Phillips and Men˜ez 1988; Les tera as having a ligneous sympodial rhizome, 4–12 vas- et al. 1997). The family is distributed mainly in the cular bundles in the cortical layer, and erect, un- temperate oceans of the northern and southern hemi- branched stems. In contrast, Zostera and Phyllospadix spheres, although the ranges of some species extend were described as having herbaceous monopodial rhi- into tropical latitudes (Hartog 1970). Contemporary zomes, two vascular bundles in the cortical layer, and taxonomic treatments of Australian Zosteraceae have a short, lateral branch at each node. Subsequent studies consistently recognized two genera: Heterozostera have questioned the taxonomic reliability of the pri- (Setch.) Hartog (monotypic as Heterozostera tasmanica mary key characters used by Hartog to distinguish (G.Martens ex Asch.) Hartog) and Zostera L., which in- Zostera and Heterozostera (Taylor 1981; Tomlinson 1982; cludes Zostera capricorni Asch., Z. mucronata Hartog, Soros-Pottruff and Posluszny 1995). When develop- and Z. muelleri Irmisch ex Asch. (Hartog 1970; Aston mental studies failed to disclose any significant differ- 1973; Robertson 1984; Phillips and Men˜ez 1988). Al- ences between the genera, Soros-Pottruff and Poslusz- though the current taxonomy of Australian Zostera- ny (1995) agreed with Taylor (1981), who recommend- ceae is widely followed, the delimitation of taxa at both ed the taxonomic reinstatement of Zostera tasmanica the generic and species levels is difficult and deserves with retention of Heterozostera as a subgenus. The same further scrutiny. Robertson (1984) observed ‘‘a broad conclusion was reached by Les et al. (1997) when a spectrum of intergrades’’ in Australian Zostera mate- molecular phylogenetic study of subclass Alismatidae rial and called for further work ‘‘to elucidate the Z. clearly placed Heterozostera within the genus Zostera.It mucronata-Z. muelleri-Z. capricorni complex.’’ is evident that the generic distinctness of Heterozostera Setchell (1933) recognized Zostera tasmanica Asch. requires further consideration. as distinct morphologically from other Zostera species The subgeneric divisions of Zostera are also in need and placed it within a monotypic section (Heterozos- of reevaluation. All Australian/New Zealand Zostera tera). Hartog (1970) later elevated section Heterozostera belong to subgenus Zosterella (Asch.) Ostenf., which to generic rank without elaboration, simply remarking Hartog (1970) distinguished by the presence of retin- that Heterozostera ‘‘. . . can easily be confused with Zos- acules, open leaf sheaths and fiber bundles in the inner

468 2002] LES ET AL.: SEAGRASS SYSTEMATICS 469 layers of the rhizome outer cortex. Because further molecular data from both nuclear and plastid com- study of these features (Jacobs and Williams 1980; partments. By determining whether classical, morpho- Tomlinson 1982) have questioned the validity of some logically defined Zostera species are supported by spe- key characters (see discussion), the taxonomic integrity cies limits indicated by molecular data, we hope to ar- of subgenera in Zostera remains unsettled. Further- rive at a taxonomic scheme for Australian Zosteraceae more, Tomlinson and Posluszny (2001) have recently that is both objective and defensible. recommended the elevation of subgenus Zosterella to generic status as Nanozostera. Clearly, the major taxo- MATERIALS AND METHODS nomic subdivisions within Zosteraceae must be recon- sidered. Morphological Analyses. Morphological characters and states The field identification of Zostera species in Austra- were compiled from pertinent publications including Aioi et al. (1998), Aston (1973), Flahault (1908), Hair et al. (1967), Harada lia continues to be difficult using the characters de- (1956), Hartog (1970), Hartog et al. (1987), Jacobs and Williams scribed in taxonomic treatments (Sainty and Jacobs (1980), Kuo and McComb (1998), Phillips and Men˜ez (1988), Rob- 1981). Taxonomic distinctions in Zostera have been at- ertson (1984), Setchell (1933), Soros-Pottruff and Posluszny (1994, tained primarily on the basis of leaf morphology (Har- 1995), Tomlinson (1982) and Yip (1988). We coded 16 vegetative and 15 reproductive characters for Phyllospadix, Heterozostera tas- tog 1970), which Phillips (1972) has shown to be en- manica,and11Zostera species (Tables 1, 2). Morphological data vironmentally labile or differentiated ecotypically were analyzed using maximum parsimony (Swofford 1998) with (Backman 1991), at least with respect to length and all character states left unordered except for character #25, which was ordered to reflect a postulated reduction series in retinacule width characters. The extent of variation in Zostera is length. Phyllospadix was used as the outgroup for rooting trees. All not adequately understood, as evidenced by recent dis- searches were conducted using branch-and bound (furthest addi- coveries of Z. caulescens having leaves an order of mag- tion sequence, MulTrees). Bootstrap support was obtained from 500 replicates. Final trees were obtained using strict consensus and nitude larger than previously reported for the species 50% majority rule consensus. The 50% majority rule consensus (Aioi et al. 1998). In particular, species recognition in tree topology was used to examine character homoplasy and to Zostera relies strongly on leaf tip and retinacule mor- provide estimates of branch lengths for identification of taxonomically significant characters. Missing data comprised 6% of morphology, which exhibit extensive variation within spe- phological data cells and were treated as missing in all phyloge- cies (Phillips and Men˜ez 1988). netic analyses. Furthermore, we have observed a number of in- Molecular Analyses. Specimens of Zostera and Heterozostera stances where critical key characters are either incon- were collected by the authors from October, 1999–January, 2000. Field collected material was immediately processed in saturated sistent with descriptions or have been improperly ap- NaCl-CTAB buffer as described by Rogstad (1992). Additional ma- plied in Zostera (see discussion). These observations terial (fresh or silica-gel dried) was provided by M. Waycott (James lead us to propose that extensive variability in key fea- Cook University, Townsville, Queensland, Australia), Y. Kadono (Kobe University, Nada, Kobe, Japan), C. T. Philbrick (Western tures (such as leaf tip morphology, nerve numbers and Connecticut State University, Danbury, Connecticut), G. Procaccini retinacule morphology), have been inadequately con- (Stazione Zoologica ‘A. Dohrn’, Naples, Italy), P. B. Tomlinson sidered in prior taxonomic keys and descriptions of (Harvard University, Petersham, Massachusetts) and Anne-Maree Schwarz (NIWA, New Zealand). Our material comprised all gen- Zosteraceae. We further suggest that historical taxo- era of Zosteraceae (Heterozostera, Nanozostera, Phyllospadix, Zostera), nomic treatments of Zosteraceae have relied on mor- six/seven species of Zostera subgenus Zosterella (including all Aus- phological concepts that do not reflect accurately the tralian species), and one/four species of Zostera subgenus Zostera variational patterns of characters used to distinguish (Table 3). Thus, all taxonomic levels of the family were represented and all Australian taxa were included. We also examined six ac- species and perhaps higher taxonomic subdivisions in cessions of Heterozostera tasmanica, four accessions of Zostera capri- the family. Thus, reconsiderations of the status of Het- corni, three accessions of Z. novazelandica, and 21 accessions of Z. erozostera, the circumscription of subgenus Zosterella, muelleri (throughout its range in Australia) to evaluate geographical divergence. and species limits in Zostera are necessary before an Routine procedures for the extraction, amplification, and se- acceptable taxonomy of these groups can be achieved. quencing of ITS (ITS-1 and ITS-2 regions including the 5.8S rDNA In this study, we evaluate taxonomic questions in gene), trnK 5Ј and 3Ј introns and rbcL followed those described in Padgett et al. (1999) and Les et al. (2002). All sequences were ob- Zostera by supplementing morphological data with tained using automated methods as described by Les et al. (2002). DNA sequence data to examine genetic variation in Due to low levels of rbcL nucleotide divergence, only one exemplar specimens of Zostera subgenus Zosterella collected for each species was compared in the analysis of rbcL sequence data. Technical difficulties (degraded DNA, failed PCR reactions), widely in Australia. By incorporating molecular mark- prevented us from obtaining trnK intron sequences for three ac- ers that are not subject to phenotypic plasticity, an in- cessions (Waycott 94018; Les 639; Les 641). We did not sequence trnK dependent test of species limits drawn from phenotyp- in two accessions (Waycott s.n.-2; Waycott s.n.-3), which represented ically labile morphological markers is provided. Uchi- populational samples of Waycott s.n.-1. Sequences used in our analyses have been deposited in the GenBank database (Table 1). yama (1996) also studied relationships of Zostera using Molecular data were analyzed using the PAUP* computer pro- molecular data (RFLP analysis of 18S ribosomal DNA), gram (Swofford 1998). Ranges in pairwise sequence divergence but that study did not include Australian taxa. We have values (uncorrected ‘p’ values) were compared among nine (ITS) and eight (trnK) Zosteraceae taxa. Gaps were treated as missing included all Zosteraceae taxa currently recognized in data in all analyses, but indel information for ITS and trnK was Australia and New Zealand and have incorporated included as separate data partitions. For each gap, different indel 470 SYSTEMATIC BOTANY [Volume 27 b ] c identical sequences within a taxon trnK, rbcL Ј b 5 trnK, (CONN); [AY077997, AY077974, Ј (CONN); [AY077996, AY077973, AY077983] (CONN); [AY077996, AY077973, AY077983, (CONN); [AY077995, AY077973, AY077983] (CONN); [AY077996, AY077973, AY077983] [ITS, 3 Voucher; Genbank accession numbers (UWA); [AY077993, n.d., n.d.] (UWA); [AY077990, AY077967, AY077978] (WCSU); [AY077985, AY077965, AY077975] (CONN); [AY077994, AY077972, AY077982] (CONN); [AY077992, AY077971, AY077981] (CONN); [AY077994, AY077972, AY077982] (CONN); [AY077994, AY077972, AY077982] (CONN); [AY077989, AY077968, AY077978] (CONN); [AY077988, AY077968, AY077978] (CONN); [AY077989, n.d., n.d.] (CONN); [AY077989, n.d., n.d.] (CONN); [AY077986, AY077966, AY077976] (CONN); [AY077991, AY077970, AY077980, AY077964] (CONN); [AY078005, AY077974, AY077984] (CONN); [AY078004, AY077974, AY077984] (CONN); [AY078003, AY077974, AY077984] (CONN); [AY078003, AY077974, AY077984] (CONN); [AY078001, AY077974, AY077984] (CONN); [AY078000, AY077974, AY077984] (CONN); [AY078002, AY077974, AY077984] (CONN); [AY077999, AY077974, AY077984] (CONN); [AY077999, AY077974, AY077984] (CONN); [AY077999, AY077974, AY077984] (CONN); [AY077998, AY077974, AY077984] (CONN); [AY077998, AY077974, AY077984] (CONN); [AY077998, AY077974, AY077984] (CONN); [AY077998, AY077974, AY077984] (CONN); [AY077998, AY077974, AY077984] (CONN); [AY077998, AY077974, AY077984, AY077962] (CONN); [AY077998, AY077974, AY077984] (CONN); [AY077987, AY077969, AY077979] (CONN); [AY078003, AY077974, AY077984] AY077984] AY077963] Schwarz s.n. Schwarz s.n. Tomlinson s.n. Procaccini s.n. Les 645 Les 644 Les s.n. Les 642 Les 641 Les 637 Les 638 Les 639 Les 636 Les 635 Les 634 Les 533 Les 536 Les 532 Les 531 Les 530 Bayer SA-99006 & Chandler Les 529 Les 528 Waycott 94018 Iida s.n. Les 626 & Jacobs 8629 Yarish s.n. Les 625 & Jacobs 8628 Les 605 & JacobsLes 8582 624 & Jacobs 8627 Waycott s.n. Waycott s.n. Waycott s.n. Waycott s.n. Waycott 94007 Les 535 Philbrick 2274 E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Њ Ј Ј Ј 53 53 10 57 57 48 14 14 27 14 47 39 47 55 12 11 44 49 50 26 51 54 45 10 59 45 00 42 45 42 42 42 13 Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ S 175 S 175 S 174 S 147 S 147 S 146 S 145 S 145 S 146 S 146 S 145 S 146 S 146 S 146 S 150 S 150 S 150 S 150 S 150 S 138 S 150 S 150 S 115 S 152 S 152 SS 145 153 S 115 SS 115 S 115 115 S 115 S 150 Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Ј Latitude/longitude 07 07 00 20 20 01 49 48 42 41 38 38 38 36 43 42 50 46 45 40 40 35 03 40 38 54 29 57 60 57 57 50 46 a Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Њ Latitude and longitude are provided for Australian and New Zealand collections; a no data. ϭ New Zealand 37 New Zealand 37 Tasmania, Australia 42 Tasmania, Australia 42 Tasmania, Australia 41 Tasmania, Australia 40 Tasmania, Australia 40 Victoria, Australia 38 Victoria, Australia 38 Victoria, Australia 38 Victoria, Australia 38 Victoria, Australia 38 Victoria, Australia 38 New South Wales, Australia 35 New South Wales, Australia 35 New South Wales, Australia 34 New South Wales, Australia 34 New South Wales, Australia 34 South Australia 34 New South Wales, Australia 34 New South Wales, Australia 32 New South Wales, Australia 30 New South Wales, Australia 30 Western Australia 34 Western AustraliaWestern AustraliaWestern Australia 31 34 34 Western Australia 31 molecular analyses. three accessions only; n.d. c Setch. New Zealand 36 Taxon Geographical origin (G.Martens ex Asch.) Hartog New South Wales, Australia 35 Asch. Queensland, Australia 16 Irmisch ex Asch. New South Wales, Australia 34 Hartog Western Australia 32 Asch. & Graebn. Hokkaido, Japan — — (Setch.) Hartog L. Connecticut, USA — — Hook. S. Watson California, USA — — Hornem. Naples, Italy — — L. Z. muelleri Z. muelleri Z. capricorni Z. muelleri Z. muelleri Z. muelleri Z. muelleri Z. muelleri Z. novazelandica Z. muelleri Z. muelleri Z. capricorni Z. muelleri Z. muelleri Z. muelleri Z. muelleri Z. novazelandica Z. muelleri Z. capicorni H. tasmanica Z. muelleri Z. novazelandica Z. muelleri Z. capricorni Z. muelleri Z. muelleri H. tasmanica H. tasmanica H. tasmanica Z. muelleri H. tasmanica H. tasmanica 1. Voucher specimens of Zosteraceae examined in Z. noltii Z. japonica Z. marina Z. mucronata P. torreyi b. c. s. r. q. p. o. n. m. l. k. j. i. h. g. f. e. d. c. b. d. b. c. d. e. f. c. b. Zostera Heterozostera Phyllospadix 7. a. 6. 5. a. 2. 3. 4. 1. a. 1. 1. a. ABLE T I) II) III) are referenced by the same accession number; 2002] LES ET AL.: SEAGRASS SYSTEMATICS 471 smooth ϭ mucronate mm) [ordered] ϭ ¾ striate; 3 persistent ϭ ϭ short (1 ϭ truncate; 3 2 mm) Ͻ ϭ ridged; 2 enclosed within spathal sheath ϭ ϭ absent or only at lowest male flower 50 deciduous; 2 ϭ Character states 36 ϭ ϭϽ This distinction made by Hartog (1970); Tomlinson (1982) and Tillich congested ( a ϭ overlap only at base crescent-shaped ϭ ϭ ϭ 150; 2 24; 3 medium (3 mm); 2 thin-walled dioecious Ͻ ϭ ϭ translucent membranous short, thick ϭ ϭ nerveless 50 soft, ephemeral notched or truncate; 2 ϭ ϭ absent absent absent absent 2 mm); 1 ϭ present terminal ϭ undulating ϭ Ͼ ϭ fused ϭ ϭ ϭ ϭ 500 partly coalesced to axis 7 ϭ ϭ 2 ϭϾ ϭ 4 16–20; 2 ϭ ϭ ϭ ϭ ϭ ϭ ϭ 150; 1 7; 1 4; 1 present; 1 long, thin; 1 linear; 1 elongated ( thick-walled; 1 present; 1 3–4; 1 open; 1 decays into wooly fibers; 1 overlap throughout; 1 present; 1 coriaceous; 1 notched; 1 monoecious; 1 lateral; 1 free; 1 emerging from spathal sheath; 1 present at all malecoriaceous; flowers; 1 1 1-nerved; 1 present; 1 long (14 mm); 1 ribbed (costate) longitudinally; 1 ovoid/ellipsoidal; 1 1500; 1 scarious; 1 absent; 1 12; 1 ϭ ϭ ϭ ϭ ϭ ϭ ϭϾ ϭ ϭ ϭ ϭ ϭϾ ϭ ϭ ϭϾ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ ϭ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ?): ϭ n m): ␮ haracters and character states used in a cladistic analysis of the Zosteraceae (see text for references). Character : a phology: phology: phology: 2. Morphological c ABLE 2 Root morphology: 3 Rhizome growth form: 4 Rhizome internodes: 1 Primary root 5 Rhizome cortical cells: 6 Rhizome cortical fiber7 bundles: Rhizome cortical vascular8 bundles (maximum): Squamule # (maximum): 9 Leaf sheath: T 10 Leaf sheath persistence: 11 Leaf sheath margins12 (at Max. maturity): leaf length13 (cm): Leaf margin ‘‘fin14 cells’’: Leaf blade texture: 17 Sexual condition: 18 Reproductive shoot position: 15 Max. #16 leaf blade Mature nerves: leaf tip: 19 Peduncle: 20 Spadix: 21 Retinacules: 22 Retinacule texture: 23 Retinacule nervature: 24 Retinacule vasculature: 31 Mature pollen length ( 25 Retinacule length (maximum): 26 Seed surface: 27 Fruit mor 28 Exocarp: 29 Fruit ‘‘grappling apparatus’’: 30 Chromosome number (2 (1995) report absence of primary root for all Zosteraceae. Vegetative mor Reproductive mor 472 SYSTEMATIC BOTANY [Volume 27 ? 1 1 1 1 1 1 1 1 1 0 1 1 variants were scored as discrete character states in the partitions. Indel data were analyzed separately for ITS, but not trnK sequenc- ? ? 0 2 0 2 0 0 2 1 3 0 0 es which had only three gaps in our alignment.

parsimony Molecular data were analyzed initially as six separate partitions: 0 0 0 0 0 0 0 0 0 1 0 0 0 1) ITS excluding gaps; 2) ITS gap data alone; 3) ITS including gap data; 4) trnK including gap data; 5) rbcL (no gaps present); 6) all 0 0 0 0 0 0 0 0 0 1 0 0 0 molecular data combined (except rbcL). Analysis of the rbcL data was provided only for comparative purposes and these data were 0 0 0 0 0 0 0 0 0 1 0 0 0 excluded from the combined molecular analyses. Trees for rbcL 27 28 29 30 31 were computed using the branch and bound algorithm. Trees for a 2 1 1 2 1 3 0 1 2 2 2 0 2 ITS, trnK and combined analyses were obtained using heuristic searches (simple addition sequence, MulTrees, TBR). Because trnK 2 2 2 2 2 2 2 0 1 data (sequence data and gaps) were invariant in all Australian/ — — — —

homoplasious in maximum New Zealand accessions of Zostera subgenus Zosterella taxa sur- 0 0 0 0 0 0 0 0 1 veyed, there were thousands of trees retained resulting in exces- — — — — sive analytical times. We resolved this problem by using only one 1 1 1 1 1 1 1 0 1 exemplar sequence from the invariant group (Les 528)inthetrnK — — — — analysis. In drawing the tree, the excluded taxa with identical se- 1 1 1 1 1 1 1 0 1 quences were added by depicting them in an unresolved ‘‘comb’’ — — — —

Three characters along with the exemplar sequence. All sequences were used in the a 0 0 0 0 0 1 1 0 0 0 0 1 1 combined molecular analyses. Missing data comprised less than 1% of data cells used in all analyses except for the combined anal- 1 1 1 1 1 1 1 1 1 0 1 1 1 ysis where they comprised 8% due to the inclusion of ﬁve accessions missing the trnK intron region. 1 1 1 1 1 1 1 1 1 0 1 1 1 Strict consensus trees were output as tree ﬁles for tree descrip- 19 20 21 22 23 24 25 26 tion purposes. Consensus trees are shown with relative branch a 0 0 0 0 0 1 1 0 0 0 1 1 1 lengths as indicated for the described trees. Bootstrap support for

state not applicable. clades was generated using 500 replicates and search parameters ϭ 0 0 0 0 0 0 0 0 0 1 0 0 0 as described above. Due to excessive analysis times, bootstrap val- 17 18 ues for the combined molecular data tree were obtaining without a 0 3 0 0 1 1 1 0 2 1 0 2 2 implementing the MulTrees option during the heuristic search. Characters 1 1 1 1 1 0 0 1 1 1 1 0 0 RESULTS 1 1 1 1 1 1 1 1 1 0 1 1 1 Morphological Analyses. Analyses using maxi- state unknown; —

ϭ mum parsimony recovered 68 trees at 45 steps with CI 1 1 1 1 1 1 1 1 1 0 1 1 1

ϭ 0.889, CI(exc) ϭ 0.792, and RI ϭ 0.857, indicating rel- 2 2 2 2 2 1 1 2 2 0 2 1 0 atively low homoplasy. The strict consensus tree (Fig.

able 2). ? 1a) was minimally resolved, but the recovered clades 0 1 1 0 1 0 1 0 0 — — — — corresponded well to taxonomic subdivisions tradi- 1 1 1 1 1 1 2 1 1 0 1 1 1 tionally and recently applied to the genus. The 50% majority rule consensus tree (Fig. 1b) was better re- 0 0 0 0 0 1 1 0 0 0 0 1 1 solved but still showed relatively weak separation of ? ? 1 1 1 1 1 1 1 1 1 0 1 species. Character analysis on this tree (using either ACCTRAN or DELTRAN) indicated only three hom- 1 1 1 1 1 1 1 1 1 1 0 1 1 oplasious characters (#16, leaf tip morphology; #18, re- 0 0 0 0 0 0 0 0 0 1 0 0 0 productive shoot position, and #26, seed surface morphology). 1 1 1 1 1 1 1 1 1 0 1 1 1 The genus Phyllospadix was clearly differentiated from all other Zosteraceae by 19 morphological char- 0 0 0 0 0 0 0 0 0 1 0 0 0 acter states (61.3% of total). Zostera tasmanica (ϭ‘‘Het- 0 0 0 0 0 0 0 0 0 0 1 0 0 erozostera’’) associated with species of Zostera subgenus Zosterella in 75% of the equally parsimonious trees re- 0 0 0 0 0 0 0 0 0 1 0 0 0

trix for Zosteraceae taxa (characters and states from T covered. The majority rule consensus tree resolved Z. 12345678910111213141516 ? 0 0 0 0 0 1 1 0 0 1 1 1 noltii and Z. japonica as a distinct group within subgenus Zosterella, but bootstrap support was weak for most species associations. Clades corresponding to previous taxonomic subdivisions were well supported erella morphologically, e.g. subgenus Zosterella (71% boot- 1.0 for the remainder. Zost Zostera strap support) and subgenus Zostera (96% bootstrap ϭ

3. Morphological data ma support). The former (ϭ ‘‘Nanozostera’’) is supported subg. subg. by two characters (#18, reproductive shoot position; ABLE

T ϭ Z. japonica Z. mucronata Z. muelleri Z. noltii Z. novazelandica Z. capensis Z. capricorni Z. asiatica Z. caespitosa Z. marina Z. caulescens #25, max. retinacule length) and the latter ( ‘‘Zostera’’ Zostera Phyllospadix Heterozostera Zostera analysis; CI sensu stricto) by three characters (#9, leaf sheath mor- 2002] LES ET AL.: SEAGRASS SYSTEMATICS 473

FIG. 1. Morphologically derived maximum parsimony cladograms for Zosteraceae. A) Strict consensus tree (branch lengths not proportional) showing poor resolution of most taxa. Previously proposed generic segregates are indicated in parentheses. B) Resolution of taxa as indicated by the 50% majority-rule consensus tree (converted into a phylogram showing proportional branch lengths). Bootstrap support (%) is indicated above branches; frequency of occurrence (%) in the majority rule tree is indicated in parentheses below branches. Numbers in square brackets indicate characters (apomorphies) from Tables 1–2 (- ϭ no characters) that define the terminal taxa on the tree. phology; #15, max. number of leaf blade nerves, #21, Four species (Z. capensis, Z. japonica, Z. muelleri, Z. retinacule occurrence). Zostera tasmanica was well-de- noltii) lacked defining character states (autapomor- fined morphologically by four, non-homoplasious char- phies) entirely. Three species (Z. caulescens, Z. mucron- acters (#3, rhizome growth form; #7, rhizome cortical ata, Z. novazelandica) were differentiated only by states vascular bundle number; #24, retinacule vasculature; of character #16 (leaf tip morphology), which was also and #30, chromosome number). one of two defining characters for Z. capricorni and Z. 474 SYSTEMATIC BOTANY [Volume 27

TABLE 4. Observed range in percent (%) nucleotide divergence (trnK—upper half; ITS—lower half) derived from uncorrected distance (‘p’) for pairwise comparisons of nine Zosteraceae taxa recognized in previous taxonomic treatments. CAP ϭ Z. capricorni;JAPϭ Z. japonica; MAR ϭ Z. marina;MUCϭ Z. mucronata; MUE ϭ Z. muelleri; NOL ϭ Z. noltii; NOV ϭ Z. novazelandica;PHYϭ Phyllospadix;TAS ϭ Z. tasmanica. N.D. ϭ no data.

NOV MUE MUC CAP NOL JAP TAS MAR PHY NOV — 0.0 N.D. 0.0 0.6 0.3 0.6–1.0 5.9 6.9 MUE 0.0–0.5 — N.D. 0.0 0.6 0.3 0.6–1.0 5.9 6.6–6.9 MUC 0.5 0.5–1.1 — N.D. N.D. N.D. N.D. N.D. N.D. CAP 0.2–0.3 0.0–1.0 0.8–1.0 — 0.6 0.3 0.6–1.0 5.9 6.6–6.9 NOL 2.2 2.0–2.7 2.9 2.2–2.5 — 0.2 1.1–1.4 6.0 7.3 JAP 2.2 2.0–2.7 2.9 2.2–2.5 1.7 — 0.9–1.2 6.2 7.3 TAS 5.4–5.8 5.5–6.3 5.9–6.3 5.4–6.3 5.7–6.0 5.8–6.1 — 6.0–6.2 6.7–8.0 MAR 16.1 16.1–16.6 16.8 16.3–16.6 16.3 16.3 16.0–16.4 — 7.4 PHY 26.0 25.8–26.5 26.7 26.0–26.3 25.2 25.7 21.8–23.9 25.6 —

marina. Single characters deﬁned Z. asiatica (#26, seed variable characters, 8 (20.0%) were informative phylo- surface morphology) and Z. caespitosa (#10, leaf genetically. Parsimony analysis produced a single tree sheath). Of these 11 species, only two non-homopla- of 41 steps with CI ϭ 0.98; CI (exc) ϭ 0.89 (excluding sious characters (#10, leaf sheath; #8, squamule num- autapomorphies); RI ϭ 0.89 (Fig. 3). Bootstrap support ber) served as deﬁning synapomorphies (for Z. caes- ranged from 63–96% for the three internal nodes re- pitosa and Z. marina respectively). solved. The rbcL topology was congruent with those Molecular Analyses. ITS DATA. Of the 640 ITS/ obtained in all other analyses. 5.8S sequence characters included in the analysis, 417 TRNK INTRON DATA. Sequence and indel data from (65.2%) were invariant; of the 223 variable characters, the two introns of the plastid trnK gene generated 929 66 (29.6%) were parsimony informative. Pairwise com- characters of which 839 (90.3%) were constant; of the parisons of sequence divergence (‘p’ distance) values 90 variable characters, 27 (30%) were informative. Par- ranged from 0–1.1% (among all Australian/New Zea- simony analysis generated a single tree of 103 steps land subgenus Zosterella taxa) to 25.6% (Phyllospadix with CI ϭ 0.97; CI (exc) ϭ 0.91 (excluding autapomor- vs. Zostera) (Table 4). Maximum parsimony analysis of phies); RI ϭ 0.93 (Fig. 4a). Bootstrap support ranged the ITS sequence data (excluding gaps) yielded 2,300 from 63–100% for nodes resolved in the analysis. The trees of 265 steps (CI ϭ 0.97; CI(exc) ϭ 0.90; RI ϭ 0.97). topology of this tree was congruent with those gen- Gaps in ITS provided 29 characters ranging from 2–5 erated from morphological, rbcL and ITS data (Figs. 1– states. Parsimony analysis of the gap data alone re- 3). sulted in a single tree (41 steps), which was devoid of COMBINED ITS/TRNK INTRON DATA. The com- homoplasy (CI ϭ 1.00; CI(exc) ϭ 1.00; RI ϭ 1.00; Fig. bined molecular data set comprised 1,598 characters 2a). Gaps were identical in all 25 accessions of Austra- where 1,256 (78.6%) were constant; of the 342 variable lian/New Zealand Zostera (excluding Z. tasmanica). characters, 106 (31.0%) were parsimony informative. Combined ITS sequence/gap data generated 669 char- Maximum parsimony analysis of these data generated acters. Of these, 417 (62.3%) were invariant; of the 252 2,301 equally minimal length trees of 410 steps (CI ϭ variable characters, 79 (31.4%) were parsimony infor- 0.97; CI(exc) ϭ 0.91; RI ϭ 0.97; Fig. 4b). Bootstrap sup- mative. Maximum parsimony analysis of the ITS se- port for nodes ranged from 61–100% (Fig. 4). quence/gap data yielded 2,300 trees at 306 steps (CI Although cladograms generated from all of the dif-

ϭ 0.97; CI(exc) ϭ 0.92; RI ϭ 0.97; Fig. 2b). ferent data sets differed by their relative degree of res- Because of polyploidy in the Australian Zosteraceae, olution, there was only one minor conﬂict between ITS we carefully examined the nuclear ITS sequences for and trnK data whereby the positions of Z. tasmanica polymorphic sites. Polymorphisms were detected only accessions from New South Wales and Western Aus- at six/223 (2.7%) of the variable sites. In most cases, tralia were exchanged (Figs. 2b, 4a). We concluded that they occurred between populations of the same pre- this high degree of congruence adequately warranted sumptive species; however, some were shared between the combination of ITS and trnK data. We did not com- presumptive species (between Z. muelleri and both Z. bine the highly conservative rbcL data which was se- capricorni and Z. novazelandica). Unique polymorphisms quenced only in exemplar accessions of each taxon were observed once in Z. mucronata (Waycott 94–018) where it was invariant in Z. capricorni, Z. mucronata, Z. and once in Z. muelleri (Bayer SA-99006 & Chandler) (see muelleri,andZ. novazelandica. Also, we did not combine discussion). the morphological data, which was compiled from de- RBCL DATA.TherbcL data provided 1,182 charac- scriptions of taxa rather than from the same specimens ters of which 1,142 (96.6%) were constant; of the 40 used in the molecular analyses (which were mainly 2002] LES ET AL.: SEAGRASS SYSTEMATICS 475

FIG. 2. Maximum parsimony cladograms for Zosteraceae derived from ITS/5.8S DNA sequence data. Latitudes for terminal clade taxa provided for comparative purposes. Australian states of origin are abbreviated as: NSW ϭ New South Wales, QLD ϭ Queensland, SA ϭ South Australia, TAS ϭ Tasmania, VIC ϭ Victoria, WA ϭ Western Australia. NZ ϭ New Zealand. USA ϭ United States. A) Single most-parsimonious phylogram (branch lengths proportional) resulting from phylogenetic analysis of indel data (sequence data excluded) with scale provided. Bootstrap support (%) for nodes is shown above branches. B) Strict consensus tree (converted into phylogram; relative branch lengths indicated by scale) based upon analysis of ITS/5.8S DNA sequence data (indel data included) showing similar resolution. Bootstrap support (%) for nodes is shown above branches. 476 SYSTEMATIC BOTANY [Volume 27

dations and considered both Heterozostera and Zostera as having monopodial, herbaceous rhizomes. Instead, she relied on differences in cortical vascular bundle number (employed as the secondary key character by Hartog) and retinacule shape to separate the genera. However, Yip (1988) showed overlap in the number of cortical bundles in Zostera (2–4) and Heterozostera (2– 12). From our experience, the number of cortical bundles recorded depends on where the section of the in- ternode is taken; apparently, leaf traces separate sooner after the node in Heterozostera than in Zostera.The FIG. 3. Single most-parsimonious phylogram (relative above studies effectively nullify the original key mor- branch lengths indicated by scale) resulting from phylogenetic phological differences used by Hartog (1970) to distin- analysis of rbcL data of nine Zosteraceae taxa. Bootstrap sup- guish Zostera and Heterozostera, therefore the generic port (%) is shown at nodes. distinctness of Heterozostera, at least as circumscribed by Hartog, cannot be accepted. vegetative and lacked many of the characters consid- Although Soros-Pottruff and Posluszny (1995) set- ered). Nevertheless, cladograms from both the mor- tled the question of rhizome construction in Heterozos- phology (Fig. 1) and rbcL (Fig. 3) data were entirely tera and Zostera (both monopodial), their clarification compatible with those of the other data sets. provided a new distinction between the taxa, i.e., an undulating growth pattern which, in the family, is ap- DISCUSSION parently unique to Heterozostera. Soros-Pottruff and For over 30 years, Hartog (1970) has been the pri- Posluszny (1995) also included the presence of wiry, mary source for seagrass identification and many au- erect stems, a tendency toward increased cortical vas- thors have faithfully adopted his taxonomy, keys and cular bundles, and lack of vascularization in retina- descriptions, usually with few modifications. However, cules as additional features that separate this taxon Hartog’s (1970) taxonomic treatment of Zosteraceae (as from other Zostera species. Three of these characters Potamogetonaceae subfamily Zosteroideae) contains nu- (#3, 7, 24, Table 2) emerged as defining apomorphies merous ambiguities and discrepancies in relevant keys in our formal cladistic analysis (Fig. 1b). Retinacule and descriptions, which have made his taxonomic morphology, which is lanceolate in Heterozostera and scheme difficult to apply. Here we wish to thoroughly triangular/suborbicular in Zostera (Roberts 1984) can reconsider that treatment with respect to the taxonomy be added as another character. According to Hartog 1 of Zosteraceae, particularly as it bears upon the delim- (1970), retinacules are elongate (2 /2-14 mm) in Phyl- itation of Australian taxa. lospadix, moderately long (2,3 mm) in Heterozostera and 1 3 Heterozostera, Nanozostera or Zostera? The generic either short ( /2-1 /4 mm) or absent in Zostera. Differ- distinction of Zostera and Heterozostera has become in- ences in retinacule length appear to be more approx- creasingly unsettled due to uncertainty in the reliabil- imate than exact. Retinacule length in ‘‘Zostera ameri- 3 3 ity of key taxonomic characters. Taxonomists have cana’’ (ϭ Z. japonica) is given as /4-1 /4 mm in the 3 1 found it difficult to separate the morphologically sim- Latin diagnosis, but as /4-1 /4 mm in the accompa- ilar Zostera and Heterozostera (Aston 1973; Jacobs and nying English description (Hartog 1970). Retinacule 1 Williams 1980). Aston (1973) and Phillips and Men˜ez length in Z. capricorni (1–1 /3 mm) is somewhat shorter 3 (1988) followed Hartog (1970) who distinguished be- than in ‘‘Z. novazelandica’’ (1–1 /4 mm; Hartog 1970) tween monopodial (former) vs. sympodial (latter) rhi- which is usually regarded as synonymous with Z. ca- zomes to separate the genera. However, Tomlinson pricorni (Phillips and Men˜ez 1988). Regardless, the lon- (1982) and Soros-Pottruff and Posluszny (1995) ger (Ն 2 mm) retinacules of Heterozostera separate it 3 showed that this often cited feature (sympodial, un- from Zostera (Ͻ1 /4 mm) without overlap. branched rhizome) is erroneous and should not be Although some distinctions between Heterozostera used to distinguish the genera. Tomlinson (1982) re- and Zostera are flawed, a modified character set (e.g., jected this feature taxonomically, observing that rhi- above), can effectively separate these taxa taxonomi- zomes in Heterozostera can appear either sympodial or cally. Furthermore, Heterozostera is hexaploid (Kuo and monopodial. Through decisive developmental studies, McComb 1998; Kuo 2001), a ploidy level unique in the Soros-Pottruff and Posluszny (1995) demonstrated that family. Thus, the principal issue is not whether Heter- the Heterozostera rhizome is clearly monopodial (as in ozostera is distinct taxonomically, but rather which tax- all other Zosteraceae), but possesses an undulating onomic rank is most appropriate given the observed growth pattern that mimics sympodial growth. Rob- differences. Purely in a taxonomic sense, the question ertson (1984) followed Tomlinson’s (1982) recommen- of distinctness must consider whether undulating rhi- 2002] LES ET AL.: SEAGRASS SYSTEMATICS 477

FIG. 4. Maximum parsimony cladograms (strict consensus trees converted into phylograms; relative branch lengths indicated by scales) for Zosteraceae derived from trnK and combined ITS/trnK data. Bootstrap support (%) for nodes is shown above branches. Abbreviations are as in Fig. 2. A) Cladogram based on analysis of trnK data. B) Cladogram based on analysis of combined ITS/trnK data. 478 SYSTEMATIC BOTANY [Volume 27 zomes, additional vascular bundles, and long, unvas- Heterozostera by performing a cladistic analysis of rbcL cularized retinacules are ‘‘adequate’’ to separate Het- sequence data for Phyllospadix, Heterozostera,andZos- erozostera and Zostera at the generic level. Obviously, tera (including species from both putative subgenera). no one answer can be defended unequivocally, as any That study firmly placed Heterozostera within a clade interpretation is subject to individual opinion. comprising Zostera species. Phylogenetic analysis of Admittedly, the circumscription of ranks (genera, rbcL data did not show Heterozostera to be distinct from sections, species) is always somewhat subjective; how- Zostera and its recognition as a genus necessarily ren- ever, greater objectivity can be achieved by adopting dered Zostera as paraphyletic. Our expanded rbcL anal- phylogenetic criteria (i.e., taxa should represent mono- ysis (which included additional taxa) produced the phyletic groups; Judd et al. 1999). Arguments concern- same result (Fig. 3). However, organismal phylogenies ing the type of characters, or number of characters nec- are more credible when inferred using multiple, con- essary to delimit a particular rank (e.g., genus) are less gruent data sets (Page and Holmes 1998). Ideally, con- compelling than those that either demonstrate or refute gruence should be observed among molecular data the monophyly of a taxon. Although several morpho- sets from different genomic compartments (e.g., nucle- logical characters once used to distinguish Heterozos- ar and plastid genomes) to avoid possible confounding tera are inappropriate, there remain unique, defining factors (e.g., paralogy, hybridization, etc.), which can features that can delimit the taxon at some rank. A create discrepancies between gene trees and species more critical question is whether recognition of Heter- trees (Soltis et al. 1998). ozostera at generic rank is defensible phylogenetically. Here we incorporated additional molecular data Alone, morphological data cannot effectively an- sets to infer the phylogenetic position of Heterozostera swer this question because of low resolution. The un- and other Zosteraceae. We analyzed DNA sequence resolved position of Heterozostera in the strict consen- data from the nuclear ITS/5.8S region of nrDNA and sus tree (Fig. 1a) is compatible with interpretations that from the trnK 3Ј,5Ј introns as well as the rbcL gene of either combine it with Zostera, or retain it as a separate cpDNA. We expanded our study to include all Austra- genus. If the topology of the majority rule consensus lian and New Zealand Zosteraceae taxa, several of tree (Fig. 1b) is used as a guideline, then Heterozostera which have been sampled from a wide range of geo- must either be combined with Zostera, or four separate graphical localities. Our results show that both nuclear genera of Zosteraceae must be recognized (see Fig. 1a) and chloroplast encoded sequences provide congruent to avoid paraphyletic taxa. phylogenetic trees for the Zosteraceae (Figs. 2–4) and The latter approach (Tomlinson and Posluszny that the tree resulting from the combined molecular 2001) seems unnecessarily excessive. Tomlinson and data (Fig. 4b) is congruent with trees derived from Posluszny (2001) proposed a new genus ‘‘Nanozostera’’ morphological data (Fig. 1). Internal support is high to accommodate species in Zostera subgenus Zosterella. for major clades in the combined molecular analysis. They provided no new data, but essentially echoed re- From these results, we conclude that molecular data sults of Soros-Pottruff and Posluszny (1995) as the ba- produce a robust and reasonable estimate of phylo- sis of their generic segregation. Because neither study genetic relationships for the taxa of Zosteraceae stud- performed a phylogenetic analysis, the conclusions ied, which is suitable for directing taxonomic decisions were reached purely on the basis of perceived mor- in this group. phological incongruities. However, our cladistic anal- Phylogenetic results using molecular data closely yses indicate that none of the genera recognized by parallel those obtained from morphology. With respect Tomlinson and Posluszny is well-defined morpholog- to DNA sequence divergence, taxa representing Zostera ically, especially when compared to Phyllospadix.‘‘Na- subg. Zosterella (‘‘Nanozostera’’) differ only slightly nozostera’’ is defined by only two synapomorphies, from Z. tasmanica (‘‘Heterozostera’’) (Ͻ 6.3%, ITS; ‘‘Zostera’’ (sensu stricto) by three synapomorphies, and Ͻ1.4%, trnK); but differ 3–5 times as much from Z. ‘‘Heterozostera’’ by four synapomorphies (see Results). marina (16.8%, ITS; 6.2%, trnK)orPhyllospadix (26.7%, In perspective, Z. noltii and Z. japonica are differenti- ITS; 7.3%, trnK) (Table 4). Because they are so weakly ated from other members of subgenus Zosterella also differentiated at both the morphological and molecular by two synapomorphies, yet have never been consid- levels, it seems superfluous to recognize either ‘‘Na- ered as a separate genus. This level of differentiation nozostera’’ or ‘‘Heterozostera’’ as separate genera. Doing is miniscule when compared to Phyllospadix, which is so would unnecessarily disrupt nomenclature in use separated from these taxa by 19 morphological apo- for more than a century while providing no useful im- morphies (Fig. 1b). Comparatively, the low level of provement to the classification. morphological differentiation would argue for the Clayton (1972) emphasized that a trend to recognize maintenance of only a single genus (Zostera) in addi- new genera in some families is slowly overtaking rec- tion to Phyllospadix. ognition of new species, a practice that blurs the dis- Les et al. (1997) tested the phylogenetic integrity of tinction between these ranks. He attributed this factor 2002] LES ET AL.: SEAGRASS SYSTEMATICS 479 to an emphasis on recognition of differences rather Our morphological cladistic analysis (Fig. 1) con- than similarities among taxa. This trend is wholly ev- firmed the utility of several characters used historically ident in Zosteraceae, where excessive generic subdi- to delimit subgenera in Zostera. Cladistically, subg. vision seems illogical, especially with so few species. Zosterella is defined by reproductive shoot position and Molecular data provide an appropriate means of dem- retinacule length, whereas subg. Zostera is supported onstrating similarity among Zosteraceae taxa, which by leaf sheath morphology, number of leaf blade should be considered along with the few morphologi- nerves, and lack of retinacules. Zostera subg. Hetero- cal differences proposed in prior taxonomic treatments. zostera is supported by its undulating rhizome, number Phylogenetic analyses of Zosteraceae resolve the of rhizome cortical vascular bundles, retinacule vas- same four clades from a variety of data, either singly culature and chromosome number. or in combination (Figs. 1–4). Although each clade Species Concept in Zostera. Zostera taxonomy has could be recognized as a distinct genus cladistically, always followed a morphological species concept. doing so would create several highly similar and Ascherson and Graebner (1907) delimited genera and weakly differentiated genera. We emphatically recom- subgenera in Zostera using reproductive characters, mend retaining only two genera in Zosteraceae, name- and determined species differences mainly by vegeta- ly Zostera and Phyllospadix, which depict the major tive characters. In their key for subg. Zosterella, species phylogenetic lineages within this family. These genera distinctions emphasized differences in leaf tip mor- are well differentiated at both the morphological and phology. Leaf tip morphology was also emphasized by molecular levels. The three subclades within Zostera Setchell (1933) in his enumeration of morphological should continue to be recognized as subgenera (see characters used to differentiate Zostera species. Setchell below), namely as subg. Zostera, subg. Heterozostera (1933, p. 812) remarked that: ‘‘. . . the tips of the ma- and subg. Zosterella (Fig. 1a). In any case, prior taxo- ture true foliage leaves are characteristic of the indi- nomic treatments recognizing Heterozostera and Zostera vidual species’’, but added that: ‘‘The differences are (incl. subgenera Zostera, Zosterella) as genera cannot be not readily expressed in words...’’ supported as a result of our phylogenetic analyses. Leaf tip morphology figured prominently in Har- Subgeneric Subdivisions in Zostera? Traditionally, tog’s (1970) keys to Zostera species, particularly for two subgenera have been recognized in Zostera: subg. subg. Zosterella where leaf tip shape appears as the Zostera (fiber bundles in outermost layers of the outer primary key character in six of the eight couplets. cortex, fused sheaths, terminal reproductive shoots, re- However, application of this character is difficult in the tinacules absent or only subtending lowest male flow- keys. Hartog’s (1970) key separates Z. capensis (leaf-tip er) and subg. Zosterella (fiber bundles in innermost lay- obtuse, deeply cleft) from Z. capricorni, Z. muelleri and ers of the outer cortex, open sheaths, lateral reproduc- Z. novazelandica (leaf-tip truncate, sometimes obtuse, tive shoots, retinacules accompanying each sta- not cleft). However, immediately following under the men)(Hartog 1970). All Australian Zostera have latter lead is Z. muelleri, which is keyed by having a traditionally been placed within subg. Zosterella (Har- leaf tip that is ‘‘obtuse or truncate, deeply notched’’, a tog 1970). combination of features thus indistinguishable from Jacobs and Williams (1980) found no difference in those of Z. capensis. Our inability to unambiguously fiber bundle distribution (Hartog’s primary key char- distinguish between ‘‘deeply cleft’’ and ‘‘deeply acter) between either Z. capricorni or Z. muelleri (subg. notched’’ makes it impossible to separate Z. capensis Zosterella)andZ. marina (subg. Zostera). Tomlinson and Z. muelleri by their leaf-tip morphology using this (1982) arrived at the same conclusion after examining key. Hartog also used leaf tip shape as the key char- a large sample of material. However, other differences acter for separating Z. capricorni (leaf-tip truncate) from exist. Retinacules occur only occasionally in subgenus Z. novazelandica (leaf-tip truncate or slightly emargin- Zostera (Hartog 1970), and the subgenera can further ate) but clearly the character states overlap making it be delimited by the fused or open condition of the leaf impossible to identify plants with truncate leaf tips. In sheath (Jacobs and Williams 1980). Similar to the case his species key for Zostera subg. Zostera, Hartog (1970) of ‘‘Heterozostera’’, segregation of subgenera in Zostera separated Z. caulescens (obtuse to mucronate leaf tips) is more reliably achieved by characters other than the from Z. asiatica (truncate to emarginate leaf tips). Yet principal key characters proposed in prior treatments. in his accompanying descriptions, the leaf tip of Z. According to Hartog’s (1970) descriptions, subgenus caulescens is characterized as ‘‘broadly obtuse,’’ which Zostera has terminal reproductive shoots (lateral in overlaps with that of Z. asiatica described as ‘‘obtuse subg. Zosterella) and seeds lacking a primary root (pri- to truncate, often emarginated.’’ mary root developed in subg. Zosterella). However, the Leaf tip morphology is no more successful in sepa- latter feature apparently represents another miscon- rating non-Australian species in Zostera subg. Zoster- ception, given that primary roots never form in Zos- ella. Hartog (1970) used leaf-tip morphology as the pri- teraceae (Tillich 1995). mary key character to distinguish Z. noltii (emarginate) 480 SYSTEMATIC BOTANY [Volume 27 from Z. japonica and Z. americana (obtuse). However, nerve number is ambiguous and should be reconsid- this distinction is contradicted in the next couplet ered as a taxonomic character. where Z. japonica is said to possess ‘‘slightly emargin- Setchell (1927) questioned the segregation of species ate’’ leaf tips and Z. americana to have ‘‘notched’’ leaf within wide ranging taxa such as Z. marina, because tips. The failure of leaf tip morphology to separate of insufficient knowledge regarding the range of mor- these three species is further evidenced by the fact that phological variation and its relationship to ecological Hartog’s ‘‘Z. americana’’ (putatively separable from Z. variation. He evaluated geographical variation in Z. japonica by leaf tip morphology) was later considered marina and a broader leaved segregate known as ‘‘Z. identical to Z. noltii (Phillips and Shaw 1976), but even- latifolia.’’ By comparing morphological variation in tually was determined to represent introduced plants specimens from the east and west coasts of North of Z. japonica (Bigley and Barreca 1982; Harrison and America, Setchell (1927) hypothesized that ‘‘Z. latifolia’’ Bigley 1982). was an environmentally induced ‘‘ecad’’ of Z. marina The number of intermediate nerves occurring be- relating to temperature differences between east vs. tween the midvein and two main lateral nerves of the west coast habitats. Setchell attributed quantitative dif- leaf blade has also been used to delimit Zostera species. ferences (length, width of leaves, # veins, # of seed coat Ascherson and Graebner (1907) distinguished Z. capri- ribs, etc.) to temperature induced differences in corni (two intermediate nerves) from Z. noltii, Z. muel- growth and development. However, in absence of ap- leri,andZ. tasmanica (intermediate nerves lacking). propriate experiments, he left open the possibility that Setchell (1933) regarded the number of intermediate these forms may represent genetically differentiated leaf nerves to be ‘‘of diagnostic importance’’ when tak- ecotypes. en ‘‘in connection with other characters.’’ Variation in Phillips (1972, 1980) concluded from reciprocal Zostera ma- nerve number comprises the first couplet in Hartog’s transplant studies that leaf morphology in rina was phenotypically plastic with respect to length (1970) key to species of subg. Zosterella. However, this and width characters often used to differentiate taxa. character is also difficult to apply. Hartog’s key to Ultimately, Phillips’ experiments led him to conclude: subg. Zosterella begins with an overlap in the primary ‘‘The use of characteristics, such as the shape of the key character (nerve number), which is described as leaf tip . . . to separate species of Zostera should be ‘‘3–5’’ in Z. capricorni and ‘‘3’’ in remaining species. discouraged’’ (Phillips and Men˜ez 1988, p. 30). It is Hartog’s description of Z. capricorni is contradictory, noteworthy that keys to Zostera species in Phillips and simply stating ‘‘nerves 5’’. Furthermore, Z. capricorni Men˜ez (1988) entirely exclude the use of leaf nerve keys out again under the group of species having only number and incorporate leaf tip morphology only with three nerves. Hartog’s (1970) key to species in subg. reservation. Backman (1991) carried out detailed com- Zostera has similar difficulties. There, the numbers of mon garden studies of Z. marina populations on the leaf nerves (7–11 in Z. caulescens; 9–13 in Z. asiatica)not west coast of North America, demonstrating the pres- only overlap, but disagree with values cited in each of ence of genetically distinct ecotypes (recognized taxo- the species descriptions (5–9 in Z. caulescens; 7–11 in nomically by him as varieties) that differed consider- Z. asiatica). In this instance, the number of nerves listed ably in their leaf morphology. The range of interspe- in the description of Z. asiatica exactly matches a key cific morphological variation in Z. marina is extensive, character for Z. caulescens. with leaves ranging from 1.5–20 mm in width (Back- We have found it difficult to determine differences man 1991). in the number of leaf nerves in specimens assigned to Although experimental investigations have not yet various Zostera taxa. Several longitudinal nerves are included Australian Zostera species, comparable levels evident under magnification in most Zostera speci- of morphological variability would be predicted. Con- mens, but the nerves differ by their degree of exsertion acher et al. (1994) observed wide morphological vari- and coloration. Specimens of ‘‘Z. muelleri’’ often have ability within populations of Z. capricorni from smooth lamina surfaces where none of the veins are Queensland, Australia, with small, medium, and large exserted, but in which three veins are darkly colored, plants occupying different regions of the littoral zone. thus rendering a tri-nerved appearance. In some spec- It would be informative to conduct similar common imensof‘‘Z. muelleri’’, there is no distinct coloration to garden experiments with Z. capricorni as those per- the veins, and some lateral veins are slightly exserted, formed by Backman (1991) for Z. marina. giving leaves a multi-nerved appearance. Specimens of Ecotypic and environmentally labile intraspecific ‘‘Z. capricorni’’ generally lack pigmentation in veins variability in width, nerve number, and tip morphol- and the lateral veins tend to be more strongly exserted, ogy, limits the taxonomic utility of these characters in thus rendering a multi-nerved appearance. However, Zostera. Presently there is no experimental evidence these categorizations are highly subjective, and often demonstrating consistent interspecific differences in differ among leaves on the same specimen. Thus, leaf these characters throughout a range of environmental 2002] LES ET AL.: SEAGRASS SYSTEMATICS 481 conditions. Until such evidence may be obtained, their that ‘‘It is not always possible to distinguish between use as taxonomic markers is not recommended. Z. mucronata and Z. muelleri with these intergrades.’’ Morphological cladistic analysis (Fig. 1b) weakly The taxonomy of Zosteraceae has been complicated separates Z. capricorni, Z. muelleri, Z. mucronata,andZ. because morphology is highly modified by ambient en- novazelandica, indicating that these taxa are distinguish- vironmental conditions. Setchell (1927) noted a corre- able only by their leaf tip morphology (character #16; lation between leaf width and water temperature in truncate, mucronate or notched) and seed surface mor- Zostera marina. Phillips (1972, 1980) demonstrated phology (character #26; striate or ridged). Both char- changes in leaf morphology associated with water acters were identified as homoplasious in our analysis, depth. Ostenfeld (1908) and Van Goor (1919) found and their practical taxonomic implementation would that Zostera marina produced narrower leaves on sand be virtually impossible due to overlap and polymor- than on muddy substrates. Environmental heteroge- phism of states (Fig. 2). We have found no other mor- neity may be responsible for some of the morpholog- phological characters that aid in the distinction of these ical variation observed in Australian Zosteraceae. taxa. ‘‘Zostera capricorni’’ is the only taxon that occurs in Hartog (1970) incorporated a secondary key char- both temperate and tropical portions of eastern Aus- acter that distinguished Z. capricorni as having rhi- tralia, regions that are characterized by different hab- zomes with two ‘‘groups’’ of roots per node, compared itats. In addition to differences in annual water tem- to other species with ‘‘2 (sometimes 1–4)’’ roots per perature, it is notable that seagrass habitats along Aus- node. It is unclear whether species with more than two tralia’s tropical east coast tend to be muddy because roots per node would possess them in two ‘‘groups’’ the Great Barrier Reef reduces wave energy, allowing or not and the character is not clarified by the descrip- fine erosional sediment to accumulate on the coast. It is also muddy where there has been intense human tions. Phillips and Men˜ez (1988) also described Z. ca- activity (clearing and erosion) thus corresponding to pricorni as having ‘‘2 groups of roots’’ per node; how- areas most accessible to botanical collectors. Seagrass ever, their illustration of this species (their Fig. 12) habitats of the southern coast are mostly sandy (Rob- clearly depicts a plant with only two roots per node. ertson 1984). Given the results of Ostenfeld (1908) and Soros-Pottruff and Posluszny (1995) determined Z. ca- Van Goor (1919) for Z. marina, it is possible that habitat pricorni to have two roots per node. Robertson (1984) differences could be responsible, at least in part, for described typical and estuarine forms of Z. muelleri some morphological differences associated with nar- which differed by having two roots per node or two rower leaved ‘‘Z. muelleri’’ phenotypes (temperate, groups of 2–6 roots per node respectively, thus indi- southern coast) and broader-leaved ‘‘Z. capricorni’’ phe- cating intraspecific variability in this character. We con- notypes (more commonly tropical, NE coast). Com- clude from these examples that Z. capricorni cannot be mon garden experiments would be informative in this distinguished by root number from other species in regard. subgenus Zosterella. Molecular data cast further doubt on the distinctness Comparisons of retinacule features in this group of species within the Australian and New Zealand also fail taxonomically as they are said to be ‘‘obliquely members of subg. Zosterella. No cpDNA variation was triangular’’ in Z. capricorni and ‘‘oblique, broadly tri- detected among accessions of Z. capricorni, Z. muelleri, angular’’ in Z. novazelandica (Hartog 1970). Under- and Z. novazelandica, which possessed identical rbcL standably, Phillips and Men˜ez (1988) merged Z. nova- and trnK intron sequences (trnK sequences were not zelandica with Z. capricorni, concluding that descrip- obtained for Z. mucronata). In contrast, trnK introns dif- tions in Hartog (1970) showed ‘‘no differences.’’ fered between accessions of these three taxa and both Some taxonomic decisions have been based on fea- Z. noltii and Z. japonica (subg. Zosterella)aswellasbe- tures perceived as unique. ‘‘Zostera mucronata’’ was tween Z. noltii and Z. japonica (Fig. 4a). Nuclear ITS/ considered distinct entirely because of its mucronate 5.8S rDNA sequences produced similar results. ITS se- leaf tip; the species was described prior to acquisition quences from all four species (including Z. mucronata) of fertile material (Hartog 1970). Yet, mucronate leaf possessed identical gaps, but their indel organization tips occur in other Zosteraceae such as Z. marina where differed from both Z. noltii and Z. japonica, which in they can be ‘‘often slightly mucronate’’ (Hartog 1970). turn also differed from each other (Fig. 2a). Minor Flahault (1908) showed that a transformation from mu- DNA sequence variation occurred within this group, cronate to indented leaf apices occurs as juvenile but mainly represented geographical discontinuities, leaves mature in Zostera noltii (as Z. nana). Further- as accessions of all four species were resolved within more, Robertson (1984) observed that: ‘‘many popu- a single clade (Fig. 2b). Our one accession of Z. mu- lations in South Australia and Victoria are now known cronata differed slightly from Z. capricorni, Z. muelleri, with some leaves bearing a more or less well-devel- and Z. novazelandica accessions (p ϭ 0.49–1.1%), but oped central mucro and other leaves notched’’ adding this level of nucleotide divergence compares to that 482 SYSTEMATIC BOTANY [Volume 27 among various Z. muelleri accessions (p ϭ 0.0–0.98%). vergence is correlated with reliable morphological Furthermore, the Z. mucronata accession was the only markers. material of this group collected from Western Austra- Finally, the influence of polyploidy must be consid- lia, thus separated from other accessions geographi- ered. The lowest reported chromosome number in Zos- cally by more than 23o longitude. Similarly, the north- teraceae is 2nϭ12 (the presumed diploid level) which ernmost accession of Z. capricorni also differed by sev- is reported for all species of subg. Zostera and for Z. eral nucleotide substitutions, yet other accessions of Z. japonica and Z. noltii in subg. Zosterella (Tables 2,3). capricorni possessed ITS sequences identical to many Southern hemisphere species (remainder of subg. Zos- Z. muelleri accessions. Geographical divergence in ITS terella)have2nϭ24 and Z. tasmanica is 2nϭ36 (Kuo was also observed for several Tasmanian and Victorian 2001). Phyllospadix (2nϭ16–20; Tables 2,3) is apparently populations of Z. muelleri (Fig. 2b). Our accessions of an aneuploid derivative of this series. Chromosomal Z. novazelandica differed by a few ITS substitutions (p data indicate that Australian/New Zealand Zostera- ϭ 0–0.49%), which probably reflects their geographical ceae represent a polyploid series derived from ances- location more than 20o longitude east of other acces- tral diploids in Zostera. The hexaploid Z. tasmanica is sions sampled. Interestingly, two collections of Z. nov- likely derived from within Zostera which contains both azelandica (Schwarz, s.n.; Table 1) were purposely sent diploid and tetraploid taxa, an interpretation consis- to us for analysis because they were ‘‘morphometri- tent with all phylogenetic analyses of Zosteraceae cally dissimilar in appearance in the field’’ (A.-M. (Figs. 1–4). Schwarz, pers. comm.). Nevertheless, despite their Wide morphological variability as observed in Aus- morphological variability, these two accessions had ITS tralian Zosteraceae is not unexpected in polyploids. As and trnK sequences identical to each other and also to Stebbins (1950) observed, polyploidy is a ‘‘complicat- the other specimen of Z. novazelandica that we exam- ing force’’ that produces ‘‘innumerable variations on ined (Tomlinson, s.n.; Table 1). old themes, but not originating any major new depar- Molecular data do not support the distinctness of Z. tures.’’ In some cases, polyploid complexes consisting capricorni, Z. mucronata, Z. muelleri,andZ. novazelandica of morphologically distinct diploids can give rise to a as discrete species, but indicate that some isolation by series of auto- and allotetraploids that span the range distance has occurred. Given that these taxa are sep- of morphological features of the diploid parents (Bayer arable morphologically essentially only by their leaf- 1999). The morphological distinctness of the diploids tip morphology (and even then with difficulty), we can be obscured by the presence of the autopolyploids, highly recommend their taxonomic merger as a single and the entire complex therefore can form a morpho- species, which, by priority, should be called Z. capri- logical continuum (Bayer 1999). Additional even-poly- corni. These results indicate that past taxonomic diffi- ploid levels, such as hexaploids, develop in mature culties in Zostera subg. Zosterella are due to recognition polyploid complexes, and these tend to be reproduc- of unnatural ‘‘species’’ that actually represent morpho- tively isolated from other ploidy levels (Bayer 1999). logical variants of a single widespread species. Because members of these ploidy levels are usually Despite the existence of considerable morphological sexual, individuals of divergent genetic makeup can variation, the similarity in reproductive features of dif- cross with ease, forming large morphological continua ferent Zostera tasmanica specimens led Hartog (1970) to within each ploidy level (Bayer 1999). Although the conclude that all material represented a single species. precise nature of polyploidy in Zostera has not been Our molecular results (Figs. 2, 4) confirm the similar- clarified, the existence of polyploidy may explain the ity of Z. tasmanica accessions, but some geographical range of morphological variability observed within molecular divergence was observed among accessions Australian Zosteraceae. of Z. tasmanica collected from different regions of Aus- Polymorphic ITS sites observed occasionally in Aus- tralia. Notably, eastern and western Australian popu- tralian section Zosterella could result from gene flow lations of Z. tasmanica exhibited slight molecular di- or polyploidy. If the former explanation is correct, then vergence in both nuclear (ITS: 1.03–1.19%) and chlo- ITS polymorphisms indicate the lack of effective iso- roplast encoded (trnK: 0.44–0.49%) DNA sequences lating barriers and the occurrence of genetic recombi- (Figs. 2b; 4a). This low level of molecular divergence nation both within and between populations regarded also probably reflects relatively prolonged geographi- previously as distinct species. On the other hand, these cal isolation of eastern and western populations of Z. polymorphisms could be ancestral remnants of an ini- tasmanica rather than evidence of a speciation event. As tial polyploid event that have homogenized differen- yet, there is insufficient evidence to warrant recogni- tially among populations by gene flow. The largest tion of any of these populations as distinct taxonomi- number of polymorphic sites occurred in Z. capricorni cally. However, a further study of eastern vs. western from northern Queensland (Les 605, Jacobs 8582), which Australian populations of Z. tasmanica could be under- was fairly remote geographically from other popula- taken to determine whether the observed molecular di- tions studied. If ITS polymorphisms resulted from 2002] LES ET AL.: SEAGRASS SYSTEMATICS 483

TABLE 5. Taxonomic scheme proposed from results of present study (* ϭ species recognized tentatively, but not evaluated in present study).

Family: Zosteraceae 1. Genus: Phyllospadix Hook. (species relationships not addressed in present study) 2. Genus: Zostera L. a. Subgenus: Zostera 1. Z. asiatica Miki* 2. Z. caespitosa Miki* 3. Z. caulescens Miki* 4. Z. marina L. b. Subgenus: Heterozostera Setch. 1. Z. tasmanica G. Martens ex Asch. c. Subgenus: Zosterella (Asch.) Ostenf. 1. Z. capensis Setch.* 2. Z. capricorni Asch. (ϭ Z. mucronata Hartog; ϭ Z. muelleri Irmisch ex Asch.; ϭ Z. novazelandica Setch.) 3. Z. japonica Asch. & Graebn. 4. Z. noltii Hornem. polyploidy, then their widespread occurrence within ASCHERSON,P.andP.GRAEBNER. 1907. IV. II. Potamogetonaceae. and between populations of section Zosterella taxa is Heft 31 in Das Pflanzenreich, ed. A. Engler. Weinheim/Bergs- trasse: Verlag von H. R. Engelmann (J. Cramer). further evidence that no effective barrier to recombi- ASTON, H. I. 1973. Aquatic plants of Australia. Carleton, Victoria, nation exists, thus supporting our contention that Australia: Melbourne University Press. these populations belong to a single, variable, wide- BACKMAN, T. W. H. 1991. Genotypic and phenotypic variability of spread species. Zostera marina on the west coast of North America. Canadian Polyploidy imposes effective reproductive isolation Journal of Botany 69: 1361–1371. (chromosomal) that may be critical for maintaining hy- BAYER, R. J. 1999. New perspectives into the evolution of polyploid complexes. Pp. 359–373 in Plant evolution in man-made habitats. drophile species where other isolating mechanisms Proceedings of the VIIth international symposium of the interna- (e.g., genetic incompatibility) are weak (Philbrick and tional organization of plant biosystematists, eds. L. W. D. van Les 1996). The relationship between polyploidy and Raamsdonk and J. C. M. den Nijs. Amsterdam: Hugo de speciation rates in aquatic plants has been pointed out Vries Laboratory. previously (Les and Philbrick 1993). Our results indi- BIGLEY,R.E.andJ.L.BARRECA. 1982. Evidence for synonymizing cate that substantial divergence exists between Zostera Zostera americana den Hartog with Zostera japonica Aschers. et Graebn. Aquatic Botany 14: 349–356. taxa of different ploidy levels, thus the clear distinction CLAYTON, W. D. 1972. Some aspects of the genus concept. Kew of Z. noltii and Z. japonica (diploids) from Z. capricorni Bulletin 27: 281–287. (tetraploid) and Z. tasmanica (hexaploid), and also the CONACHER, C. A., I. R. POINTER, and M. O. DONOHUE. 1994. Mor- distinction between all Zostera (xϭ6) and Phyllospadix phology, flowering and seed production of Zostera capricorni (xϭ4–5). Aschers. in subtropical Australia. Aquatic Botany 49: 33–46. FLAHAULT, C. 1908. 4. Gattung. Zostera L. Pp. 516–529 in Lebens- Our systematic studies warrant a revised taxonomy geschichte der Blu¨tenpflanzen Mitteleuropas. Spezielle O¨ kologie der of Zosteraceae which better reflects phylogenetic re- Blu¨tenpflanzen Deutschlands, O¨ sterreichs und der Schweiz. Vol. 1 sults obtained from a variety of molecular and non- part 1: Allgemeines, Gymnospermae, Typhaceae, Sparganiaceae, Po- molecular data (Table 5). We recognize two genera, tamogetonaceae, Najadaceae, Juncaginaceae, Alismaceae, Butoma- Zostera and Phyllospadix. Zostera is circumscribed as ceae, Hydrocharitaceae, eds. O. von Kirchner, E. Loew, and C. consisting of three subgenera, Zostera, Heterozostera, Schro¨ter. Stuttgart: Eugen Ulmer. GOOR,A.C.J.VAN. 1919. Het zeegrass (Zostera marina L.) en zijn and Zosterella, with four, one, and four species, respec- beteekenis voor het leven der visschen. Rapport Verhandelin- tively. Eventually we hope to obtain molecular data for gen Rijksinstituut Visserijonderz 1: 415–498. Z. capensis, Z. asiatica, Z. caespitosa,andZ. caulescens to HAIR,J.B.,E.J.BEUZENBERG, and B. PEARSON. 1967. Contributions further supplement this work and welcome receipt of to a chromosome atlas of the New Zealand flora. New Zea- material of any of these taxa. land Journal of Botany 5: 185–196. HARADA, I. 1956. Cytological studies in the Helobiae. I. Chromo- ACKNOWLEDGEMENTS. We gratefully acknowledge various some idiograms and a list of chromosome numbers in seven support from CIES (Fulbright Program), CSIRO (Canberra), RBG families. Cytologia 21: 306–328. (Sydney), and NSF DEB-9806537. We thank M. Waycott, Y. Ka- HARRISON,P.G.andR.E.BIGLEY. 1982. The recent introduction dono, C. T. Philbrick, G. Procaccini, P. B. Tomlinson, and A.-M. of the seagrass Zostera japonica to the Pacific coast of North Schwarz for providing us with specimens. Field and lab assistance America. Canadian Journal of Fisheries and Aquatic Sciences was provided by G. Chandler and N. Bagnall. 39: 1642–1648. 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