Systematics of the Hildenbrandiales (Rhodophyta): Gene Sequence and Morphometric Analyses of Global Collections1
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J. Phycol. 39, 409–422 (2003) SYSTEMATICS OF THE HILDENBRANDIALES (RHODOPHYTA): GENE SEQUENCE AND MORPHOMETRIC ANALYSES OF GLOBAL COLLECTIONS1 Alison R. Sherwood2 and Robert G. Sheath3 College of Biological Science, University of Guelph, Guelph, Ontario, Canada, NIG 2WI Fifty-seven collections of marine and freshwater and Hildenbrandia, which is globally distributed in both Hildenbrandia from North America, South America, marine and freshwater habitats (Rosenvinge 1917, Europe, and Africa were compared with 21 type and Bourrelly 1955, Silva et al. 1996). Hildenbrandia is a historically important specimens using multivariate crustose red alga, whereas Apophlaea possesses upright morphometrics. Additionally, phylogenetic analyses thallus portions in addition to a crustose basal thallus of 48 specimens of Hildenbrandia and two specimens (Rosenvinge 1917, Hawkes 1983). of Apophlaea were carried out based on sequences of The Hildenbrandiales has traditionally been plagued the rbcL chloroplast gene and the nuclear 18S rRNA with taxonomic problems due to a proliferation of gene. Morphometric analyses based on vegetative cell taxonomic names where few morphological charac- and filament dimensions distinguished two groups of ters are available for their separation. Several charac- freshwater Hildenbrandia specimens, the first corre- ters commonly used within the genus Hildenbrandia sponding to those collections from North America and for taxonomic purposes, such as thallus thickness and the Philippines and the second to those from Europe conceptacle dimensions (in marine species), are known and the Canary Islands. The first group had smaller to vary with the age of the plant (Pueschel 1982). In mean cell and filament dimensions (cells 4.0 ϫ 4.4 m, addition, cellular dimensions are variable in different filaments 46.5 m) and corresponded to H. angolen- parts of the thallus due to the branching filaments sis, whereas the second group had larger mean di- composing the crust (Starmach 1969), and the mea- mensions (cells 5.8 ϫ 6.6 m, filaments 55.3 m) surement of this character must be combined with rep- and represented H. rivularis. Marine specimens were resentative sampling of cells along the lengths of the morphometrically distinguishable into two groups based filaments. Although differences in these characters are on tetrasporangial division pattern as well as other evident for several species, such as the reportedly thallus characters. However, measurements and char- thicker thalli of H. lecannellieri and H. occidentalis (Har- acter determinations of some type specimens dif- iot in Askenasy 1888, Gardner 1917), they must be in- fered greatly from the original descriptions, and thus terpreted with caution, given the variation present in further work to determine the stability of these char- these characters over the life of the alga. Other char- acters is required. Phylogenetic reconstruction based acters of doubtful validity (such as the presence of para- on the 18S rRNA gene and rbcL gene sequence data physes to separate H. dawsonii from H. canariensis and generally demonstrated separation of the marine and H. crouanii) have been used previously to distinguish freshwater forms of Hildenbrandia, with some marine species (Hollenberg 1971). Reports of paraphyses are taxa forming monophyletic groups (e.g. H. lecannel- common in the Hildenbrandia literature (Hariot in lieri and H. occidentalis) and others forming para- Askenasy 1888, Womersley 1994), and their interpre- phyletic groups (e.g. H. rubra). The two specimens tation has been much debated. For example, the pres- of Apophlaea formed a monophyletic group within ence or absence of paraphyses was used, in part, to de- the paraphyletic genus Hildenbrandia. limit sections within the genus Hildenbrandia by J. Agardh Key index words: 18S rRNA gene; Apophlaea; Hilden- (1852), whereas more recent investigators have sug- brandia; Hildenbrandiales; morphometrics; rbcL gene; gested that reports of paraphyses are actually empty systematics sporangial walls in the conceptacle or fungal filaments (Ardré 1959, Denizot 1968). Pueschel (1982) provided ultrastructural evidence to support the view that they The red algal order Hildenbrandiales contains two are empty sporangial walls. genera: Apophlaea, which is limited in distribution to Despite the widespread distribution of Hildenbran- the marine coastlines of New Zealand (Hawkes 1983), dia, little work has been conducted until now to clarify the systematics of the Hildenbrandiales by comparing results from modern molecular analyses with those from traditional morphometric analyses of type specimens. The present study uses a global taxon sampling ap- 1Received 7 March 2001. Accepted 26 December 2002. proach within members of this order to compare phy- 2Present address: Department of Botany, 3190 Maile Way, University logenetic relationships based on molecular data to of Hawaii at Manoa, Honolulu, HI 96822, USA. 3Author for correspondence and present address: Provost’s Office, phenetic relationships based on morphometric data. California State University San Marcos, San Marcos, CA 92096-0001, Such an approach is necessary with this order because USA. E-mail: [email protected]. the taxonomy of the group, until now, has been based 409 410 ALISON R. SHERWOOD AND ROBERT G. SHEATH on the few morphological characters available, and gia were made, and tetrasporangial division pattern noted for quite likely the evolutionary relationships within the or- comparison with Hildenbrandia specimens. Samples were fixed in 2.5% CaCO3-buffered glutaraldehyde der will not be elucidated using these techniques alone. to prevent morphological distortion, or rehydrated after pres- Current systematic treatments in phycology generally ervation in silica gel, and were analyzed to determine associa- use a combination of morphological and molecular tions based on similarity. The following characters were mea- analyses to obtain support for taxonomic decisions from sured or determined for all collections as well as type and ϭ several sources (Saunders et al. 1995 [Acrochaetiales- historically significant specimens: cell diameter (n 30), cell length (n ϭ 30), filament length (n ϭ 30), and basal layer Palmariales complex], Vis and Sheath 1998 [Batrachos- height (n ϭ 30). Where possible, marine collections were mea- permum], Bailey 1999 [Corallinaceae], Pueschel et al. sured for the following reproductive characters: maximum con- 2000 [Audouinella]), as is used in the current study. ceptacle depth and diameter (n ϭ 10) and maximum tetraspo- The biogeography and systematics of Hildenbrandia rangial length and diameter (n ϭ 10). Presence (1) or absence (0) of protuberances on the thallus surface and the tetraspo- in both North America and Europe have been previ- rangial division pattern for each sample were also noted. Tet- ously examined (Sherwood and Sheath 1999, 2000). In rasporangial division pattern was coded as follows: divisions paral- this study we expand upon these analyses with the in- lel (1) or some divisions not parallel (0). To avoid excessive clusion of collections of the Hildenbrandiales represent- influence on the analyses by very large or very small measure- ing taxa not previously available for analysis, including ments, the data were ranged according to Dunn and Everitt (1982), which standardized the data so that quantitative mea- H. lecannellieri, H. patula, H. dawsonii, and Apophlaea sin- surements ranged between 0 and 1. For each of the following clairii. As well, collections from additional global re- analyses, both cluster analysis (UPGMA algorithm) and ordina- gions are included from such diverse locations as the tion (principal coordinates [PCO] analysis or principal compo- Philippines, Australia, the Canary Islands, Chile, Uru- nents [PCA] analysis) were performed on the data (however, only the cluster dendrograms are shown because both analyses guay, and South Africa. DNA sequence analyses of the revealed very similar trends): rbcL and 18S rRNA genes are presented here with phylo- genetic reconstruction based on single gene and com- • Analysis 1: All marine specimens for which reproductive data were available. PCO was the ordination method bined data sets, as well as morphometric analyses of used in this instance because both quantitative and qual- global collections and the type specimens. itative data were both present in the data matrix. The Gower similarity coefficient (Gower 1971) was used be- cause it allows the incorporation of both data forms. materials and methods • Analysis 2: All freshwater specimens. PCA was used here Type specimens, historically significant specimens, and global collec- because all data were quantitative. tions analyzed. Type and historically significant specimens of Data were compared using Euclidean distances. the order Hildenbrandiales (Hildenbrandia and Apophlaea) were Significance of groups resulting from the analyses was tested obtained and morphometrically analyzed (Table 1). The fol- using one-way analysis of variance (P Ͻ 0.05). Cluster and ordi- lowing type specimens were requested but were unavailable for nation analyses were carried out using MVSP 3.0 (Multi-Variate examination (herbaria from which specimens were requested Statistical Package; Kovach Computing Services 1986–1998), and are given): analysis of variance were carried out using Minitab (Ryan et al. 1. Hildenbrandia (“Hildenbrandtia”) arracana (“Arracana”) G. 1985). Measurements of the type and historically significant speci- Zeller (1873:192);