J. Phycol. 24, 377-385 (1988)

A PRELIMINARY INVESTIGATION OF THE PHYLOGENETIC RELATIONSHIPS AMONG THE FRESHWATER, APICAL PORE FIELD-BEARING CYMBELLOID AND GOMPHONEMOID (BACILLARIOPHYCEAE)

John P. KocioEek2 and Eugene F. Stoerrner Great Lakes Research Division, 2200 Bonisteel Boulevard, The University of Michigan, Ann Arbor, Michigan 48 109

ABSTRACT shape and most assume a stalked growth habit via Relationships among the apical pore jeld-bearing dia- mucilage exuded through apical pore fields (APF’s). tom genera Brebissonia, Cymbella, Gomphonema, Gomphoneis, Didymosphenia, Gomphocymbella, CLADISTIC ANALYSIS and Reimeria and their close ally Encyonema were Terminal taxa. Included in this group of diatoms evaluated using cladistics. These biraphid genera are a are those taxa which are asymmetrical to the trans- polythetic group dejned by number and type, apical axis (Gomphonema Ehrenb., Gomphoneis Cleve, mode of , valve orientation and pres- Didymosphenia M. Schm.), asymmetrical to the trans- ence of apical porejelds. Character states of valve as well apical and apical axes (Gomphocymbella 0. Mull.) and as non-siliceousfeatures were described and then polarized those taxa asymmetrical to the apical axis. Members using the outgroup method, with the naviculoid genera of this latter group had previously been included in Anomoeoneis and Placoneis sewing as outgroups. The three genera, based on differences in growth habit. cladistic analysis suggests two groups of terminal taxa, Cocconema Ehrenb. represented those cymbelloid corresponding to to a predominantly cymbelloid lineage diatoms which grow at the ends of mucilaginous and a goniphonemoid lineage. Didyrnosphenia, previ- stalks, whereas Encyonema Kiitz. species grow within ously thought to be closely allied to gomphonemoid diatoms, mucilaginous tubes. The genus Cymbella C.A. Ag. is shown to be more closely related to Cymbella. Gom- originally contained free-living species. These three phonema appears to be without distinguishing charac- genera were combined into Cymbella by Heiberg teristics. Relationships of the other genera are described. (1863) who argued that growth habit alone was not The systematic position of small, doubly-punctate Gom- sufficient to base taxonomic differences at the genus phonema species is discussed. level. This view predominated until recently when Krammer (1982b) showed the stalked forms (which Key index words: Bacillariophyeae; Brebissonia; cla- have nomenclatural priority to the name Cymbella) distics; Cymbella; diatoms; Didymosphenia; Encyo- differ substantially from those species which grow nema; evolution; Gomphocymbella; Gomphoneis; in tubes. Krammer (198213) recognized Cymbella and Gomphonema; phylogeny; Reimeria Encjonema as distinct groups (subgenera of Cymbella); they are treated here at the level of genus. The Although diatoms have been observed with light remaining cymbelloid diatoms that fit neither into microscopy for over 275 years (Anonymous 1703, Cymbella nor Encyonema constitute a heterogenous Leeuwenhoek 1703) and with electron microscopy group, called Cymbopleura by Krammer (1982b). Lit- for over 50 years (Krause 1936), little is known about tle is known about their ultrastructure. From this genealogical relationships in some groups, particu- heterogenous group of cymbelloid diatoms Kociolek larly at the generic level. Many authors have inves- and Stoermer (1987) erected the genus Reirneria, tigated relationships at this level of hierarchy; how- which includes the former Cymbella sinuata Greg. ever, more recent works have concentrated almost and its allies. Thus, of the cymbelloid diatoms, Cym- exclusively on marine and freshwater centric dia- bella sensu stricto, Encyonema and Reimeria are rec- toms. ognized as natural groups and included as terminal Relationships among pennate genera have taxa in the analysis. only recently been explored through the cladistic Also included in the analysis is the genus Brebis- approach. Williams (1985) based his view of rela- Sonia Grun., a small group described originally as tionships among members of the family Diatoma- having naviculoid symmetry (Grunow 1860) but re- ceae on results of a cladistic analysis, and Kociolek cently reported to be slightly asymmetrical to the and Stoermer (1986a) used cladistics to evaluate re- apical axis (Mahoney and Reimer 1986). lationships among monoraphid diatoms. In this re- Diagrammatic interpretations of the eight genera port we attempt to evaluate phylogenetic relation- included in the cladistic analysis are presented in ships in one group of freshwater biraphid diatoms. Figure 1. Members of this group exhibit asymmetry in valve Two of the genera, Cymbella and Gomphoneis, are subdivided. The subdivisions include: (1) Cymbella

I Accepted: 6 May 1988. mexicana (Ehrenb.) Cleve separated from Cymbella S. Address for reprint requests. s. based on Krammer’s (1982b) observations of dif- 377 378 JOHN P. KOCIOLEK AND EUGENE F. STOERMER

BREBISSONIA ities between most species with regard to pattern of reduction division and sexual reproduction. Mann (1983a) observed that the genera Cymbella s. l., Gom- phonema and Didymosphenia, among others, produce ENCYONEMA valves of cis orientation only, and similar observa- tions have been made for Gomphoneis (Kociolek 1988). This provides further evidence that these genera are closely related. Dawson (1973b, c), COX (1977), Kociolek and Rosen (1984), Mahoney and Reimer (1986) and Kociolek and Stoermer (1987) have noted similarities between most of these genera based on APF structure. Representatives from all genera except Encyonema form mucilaginous stalks. REIMERIA Thus, similarities of chloroplast shape, sexual re- production, and APF structure and function suggest these genera form a monophyletic lineage. GOMPHOCYMBELLA Previous interpretations of phylogenetic relationships. - Previous classification schemes for the cymbelloid ? e and gomphonemoid genera considered here have traditionally been based on valve symmetry. Cym- MOSPHENIA belloid diatoms were previously classified together, and gomphonemoid diatoms were thought to form a separate natural group, based on their symmetry features (Muller 1905, Karsten 1928, Patrick and Reimer 1975). The genus Brebissonia has occupied a primitive place in this group, owing to its navic- uloid symmetry, whereas Gomphocymbella, which is asymmetrical to both the apical and transapical axes, has been suggested to be intermediate between cym- belloid and gomphonemoid forms. Very little evidence, other than symmetry consid- FIG. 1. Schematic representation of valve (left) and cytological erations, has been offered to suggest phylogenetic (right) features of the eight genera serving as terminal taxa in relationships between the cymbelloid and gompho- the cladistic analysis. At right, dark circles represent nuclei, dot- nemoid diatoms. Cleve (1894) and Yermoloff (19 18) ted circles represent . pointed to members of the genus Navicula Bory as possible ancestors of the genus Cymbella. The pos- sibility of Navicula-like progenitors to the cymbel- ferences in the structure of foramina, stigmata and loid diatoms has been strengthened by the work of APF’s, and (2) separation of Gomphoneis into two Cox (1979) and Krammer (1982b) which shows at- groups, namely G. elegans (Grun.) Cleve and its allies tributes of the Navicula lineolatae group in cymbel- and G. herculeana and its allies. Distinction between loid diatoms. Mann (1983b) has also suggested the the two groups represents those species without and possible origin of cymbelloid forms from Navicula. with an axial plate, respectively. Some authors have suggested that gomphonemoid A complete listing of terminal taxa is presented diatoms may have arisen from the cymbelloids. Cleve in Table 1. (1894) indicates that gomphocymbelloid diatoms may The freshwater APF-bearing cymbelloid and gompho- be intermediate between the two groups, while nemoid diatoms and their close allies as a monophyletic Krammer and Lange-Bertalot (1985:38) state that lineage. The hypothesis that the terminal taxa form Reimeria sinuata (Greg.) Kociolek & Stoermer is, a monophyletic group is suggested by cytoplasmic, “eine(r) transitorische(n) Art zwischen Cymbella und reproductive, valve and growth habit characteris- Gomphonema. ” tics. As early as 1871 Pfitzer noted similarities in Characters and character polarity. Characters used cytoplasmic features and modes of reproduction be- in the cladistic analysis are described below. Defi- tween species of Cymbella s.1. and Gomphonema. Mer- nitions and distribution of character states are taken eschkowsky (1902- 1903) grouped most of these largely from previously published observations. Po- genera together based on similarities of larity of character states was determined by the out- and, although Gomphoneis was not included in Mer- group method. This criterion for establishing char- eschkowsky’s treatment, unpublished observations acter polarity assumes features found within the indicate G. herculeana has a similarly-shaped chlo- group under investigation (the ingroup) and in sister roplast (Kociolek 1988). Hauptfleisch (1 895), Stein- taxa (the outgroups) were also found in the ancestor ecke (193 l), and Geitler (1973) have noted similar- of the groups and therefore are primitive. Features PHYLOGENETIC RELATIONSHIPS OF DIATOMS 379

TABLE1. Coding of character statesfor the ten terminal taxa. 0 = primitive, I, 2 = derived, 9 = missinglunknown. * represents characters with unordered states. See text for description of features.

Tam

~~ DtdpOS- Gom ho Gomphoneu Features Brebrrsonia Cymbella C. mencana phenta Encyonema cym&ll~ elegnns hernrleana Gomphonema Rnmena

1. 0 1 1 1 1 1 0 0 0 1 2. 1 0 0 1 0 1 1 1 1 0 3. 0 0 0 1 0 1 1 1 1 0 4. 0 0 0 1 0 1 1 1 1 0 5. 0 1 1 1 0 0 0 0 0 0 6. 1 0 0 0 0 1 0 1 1 0 7. 0 1 0 0 0 0 0 0 0 0 8. 0 0 1 1 0 0 0 0 0 0 9. 0 0 1 1 0 0 0 0 0 0 10. 0 1 1 1 0 1 0 1 1 0 11. 0 0 0 0 0 0 0 0 0 1 12. 0 0 0 0 0 0 1 1 0 0 13. 0 0 0 0 0 0 1 1 0 0 14. 0 0 0 0 0 0 1 1 0 0 15. 0 0 0 0 0 0 0 1 0 0 16. 0 1 1 1 0 0 0 0 0 0 17.* 9 2 2 2 1 1 9 1 1 1 18.* 9 2 2 2 1 1 9 1 1 1 19.* 9 2 2 2 1 9 9 1 1 1 20. 0 1 1 0 1 0 0 0 0 0 21. 1 1 1 1 0 1 1 1 1 1 found in ingroup taxa only are considered derived, sents lack of an intermissio. Most naviculoid dia- having evolved after the branching event that led toms, including the outgroups, have an intermissio, to the ingroup and outgroup taxa (Watrous and and its absence is considered derived. Wheeler 1981, Maddison et al. 1984, Kociolek 1986). 6. Internal stigmal openings slit-like. An unoccluded, The naviculoid genera Placoneis Meresch. emend Cox unspecialized pore in the central area is considered and Anomoeoneis Pfitzer serve as outgroup taxa in primitive, because such a condition is observed in this analysis. A close relationship between the out- Placoneis (Cox 1987) and certain members of Ano- groups and the terminal taxa is suggested by simi- moeoneis (unpubl. observ.). This feature is also found larities in chromatophore structure (Schmidt 1876, in Gomphoneis elegans (Kociolek and Stoermer 1988). Mereschkowsky 1902-1903, Heinzerling 1908) and Any elaboration of the internal opening, such as the the fact that species of both outgroups may possess slit-like stigmal openings found in Brebissonia (Ma- isolated puncta in the central area (Patrick and Rei- honey and Reimer 1986), Gomphonema (Dawson mer 1975, Cox 1987), a feature found in most in- 1973c), Gomphocymbella beccari (Grun.) Forti (Kram- group taxa. mer 1982a), and species closely allied to Gomphoneis 1. Asymmetry about the apical axis. This character is hercuteana (Kociolek and Stoermer 1986b, c) is con- found in Cymbella, C. mexicana, Gomphocymbella and sidered derived. species of Didymosphenia (Schmidt 1899, Skvortzow 7. Dendritic stigmata. Cymbella species exhibit a den- and Meyer 1928, Hustedt 1930). Lack of this feature dritic overgrowth of the stigmal opening (Cox 1977, in the outgroups suggests its presence is derived. Krammer 1982b, Schoeman and Ashton 1983), and 2. Asymmetry about the transapical axis. This feature this is considered derived relative to its absence in is found in Didymosphenia, Gomphonema, Gomphoneis the outgroups. species, and Gomphocymbella but is lacking in the out- 8. Stigmata covered by silica. In C. mexicana (Kram- groups. Its presence is therefore considered derived. mer 1982b) and Didymosphenia (Dawson 1973a, b) 3. Asymmetry about the pervalvar plane. This feature the internal opening of the stigma is raised and con- appears to be associated with asymmetry about the ical with a siliceous covering. This stigma feature is transapical axis. It is lacking in the outgroups, sug- considered derived since this feature is absent in the gesting its presence is derived. outgroups. 4. APF restricted to one pole. This feature is corre- 9. Foramina complex. The ornate, external siliceous lated with both [2] and [3] and is considered derived structures of the foramina found in C. mexicana relative to a symmetrical positioning of porelli at (Krammer 198213) and Didymosphenia (Okuno 1954, both poles. Desikachary 1957, Dawson 1973a, b) are considered 5. Intermissio lacking. As illustrated by John (1982) derived relative to the unoccluded puncta found in and Krammer (1 982b: pl. lOSl), continuation of the the outgroups. The possible significance of C-shaped internal raphe fissure across the central area, ac- foramina, as well as the number of rows of puncta companied by a hooded siliceous structure, repre- composing the striae, has been discussed by several 380 JOHN P. KOCIOLEK AND EUGENE F. STOERMER investigators (Cleve 1894, Okuno 1974, Mann sible for the image of longitudinal lines in the G. 1981b, Kociolek and Rosen 1984, Krammer and herculeana subgroup (Kociolek and Rosen 1984, Ko- Lange-Bertalot 1985). Recent observations, how- ciolek and Stoermer 1986b, c, Kociolek et al. 1986). ever, suggest the type of puncta produced may vary None of the other terminal taxa or the outgroups within genera and perhaps even within a species possesses this feature, thus its presence is considered (Krammer 1982a, Krammer and Lange-Bertalot derived. 1985, Kociolek and Stoermer 1987). Also Krammer 16. APF not bisected by external raphe ends. In most and Lange-Bertalot (1985) have suggested C-shaped of the terminal taxa, as well as the outgroups, the foramina and doubly-punctate striae may be ho- raphe bisects the terminal group of pores. In most mologous (but see Kociolek and Stoermer 1987). Cymbella species (Krammer 1979, 1981, 1982b) as Due to the confusion over this feature(s?) we have well as C. mexicana (Krammer 1982b) and Didymo- avoided using presence of C-shaped foramina or sphenia (Dawson 1973b, c) the distal raphe ends curve number of punctum rows in the striae as features. abruptly and do not bisect the APF’s. The latter 10. Differentiated APF’s. In most of the APF-bear- condition is considered derived. ing taxa, porelli of the APF’s are physically sepa- 17. Placement of stigmata or isolated pores. Stigmata rated from the striae by an unornamented strip of are placed along the ventral margin in Cymbella, C. silica and structurally differentiated from the puncta mexicana (Krammer 1982b) and Didymosphenia (Mul- (Dawson 1972, 1973a, b, c, Hufford and Collins ler 1905). In the latter genus this condition is most 1972a, b, c, Schrader 1973, Gerloff and Helmcke evident in species asymmetrical to the apical axis [D. 1977, Krammer 1979, 198213, Kociolek and Stoer- curuirostrata (Temp. & Brun) M. Schm., D. sibirica mer 1986~).The alternative condition, that of un- (Grun.) M. Schm.; Schmidt 1899, Skvortzow and differentiated porelli that appear as condensed striae, Meyer 19281. Stigmata or isolated pores are placed is found in Brebissonia (Mahoney and Reimer 1986), along the dorsal margin in Encyonema (Krammer Gomphoneis elegans (Grunow in Van Heurck 1880, 1982b, Mann 1983b), Reimeria (Reimer in Patrick Patrick in Patrick and Reimer 1975, Kociolek and and Reimer 1975, Schoeman and Archibald 1978) Stoermer 1988) and Reimeria (Kociolek and Stoer- and Gomphocymbella (Fricke 1902, Miiller 1905, Hus- mer 1987). The outgroup taxa do not have differ- tedt 1930). That stigmata are placed dorsally in Gom- entiated pores at the poles, thus differentiated po- phonema and Gomphoneis herculeana is evidenced by relli are considered derived. specimens asymmetric to the apical axis (Muller 1905, 11. Unequal APF’s. The unequal distribution of Mayer 1928; pers. observ.) that have stigmata placed APF porelli at the poles of Reimeria (Kociolek and along the more highly arched (dorsal) margin. The Stoermer 1987) appears to be unique both within condition in G. elegans and Brebissonia, which have the ingroup and among the outgroup taxa; there- stigmata on either side of the central area, cannot fore, unequal APF’s are considered derived. be determined. Because these conditions are lacking 12. Septa present. Members of the genus Gompho- (or at least cannot be determined) in the outgroups, neis possess a septum at the headpole and the foot- the dorsal (a) and ventral (b) positions are each con- pole (Schmidt 1899, Kociolek and Rosen 1984, sidered derived and unordered. Krammer and Lange-Bertalot 1985, Kociolek and 18. Dejection of external distal raphe ends. Two con- Stoermer 1986b, c, Kociolek et al. 1986); lack of ditions exist for the terminal taxa. One group of septa in the outgroups suggests their presence is taxa (Cymbella, C. mexicana, and Didymosphenia) pos- derived. sess distal raphe ends that are deflected dorsally. 13. Pseudosepta present. Pseudosepta have been re- The second condition, ventrally deflected raphe ported and illustrated at the headpole and footpole ends, is found in Encyonema (Krammer 1982b), Rei- of Gomphoneis species (Schmidt 1899, Kociolek and meria (Kociolek and Stoermer 1987) and Gompho- Rosen 1984, Kociolek and Stoermer 1986b, c, Ko- cymbella (Krammer 1982a), as well as Gomphonema ciolek et al. 1986). The outgroups lack these fea- and Gomphoneis herculeana as determined in [ 171. tures, suggesting their presence is derived. The condition for G. elegans and Brebissonia cannot 14.Marginal laminae present. The internal siliceous be determined. Because both ventrally deflected (a) covering along the valve mantle and margin that and dorsally deflected (b) raphe ends are lacking in coalesces with interstriae before reaching the girdle the outgroups (or cannot be determined), both states bands is known as the marginal laminae. They are are considered derived and unordered. present in species of the G. herculeana subgroup (Ko- 19. Position of the chloroplast, and nucleus. ciolek and Stoermer 1986b, Kociolek et al. 1986, This feature has been discussed relative to the seg- Kociolek 1988) and G. elegans subgroup (Krammer regation of Encyonema and Cymbella species (Geitler 1982a, Krammer and Lange-Bertalot 1985, Koci- 1958, 1981, Mann 1981a). Species of Cymbella and olek and Stoermer 1988). This feature is lacking in C. mexicana possess a dorsal chloroplast with a dorsal the outgroups, which suggests its presence is de- pyrenoid and ventral nucleus (Cholnoky 1929, Geit- rived. ler 198 1). A ventrally rotated chloroplast with ven- 15.Axial plate present. This internal siliceous struc- tral pyrenoid and dorsal nucleus is observed in En- ture is expanded from the axial area and is respon- cyonema (Cholnoky 1929, Geitler 1981, Mann 1983b) PHYLOGENETIC RELATIONSHIPS OF DIATOMS 38 1

- Q)

Sexual reproduction: Geitler type IAlaor

Recurved internal prox. raphe ends

Isolated puncta

FIG. 2. Consensus cladogram of the three equally most parsimonious cladograms constructed by PAUP for the character state data presented in Table 1. Horizontal black bars represent shared, derived features. Numbers correspond to features described in the text and Table 1. “X” indicates evolutionary reversals. and Reimeria (Geitler 1958). The dorsal stigma in MATERIALS AND METHODS Gomphonema species (see [IS]) is positioned opposite Coding of the character states for the ten terminal taxa is the pyrenoid (Dawson 1972) and a similar condition presented in Table 1. Three most parsimonious cladograms were is observed in Gomphoneis herculeana (Kociolek 1988). identified by PAUP (Phylogenetic Analysis Using Parsimony, Thus, these two taxa have ventral chloroplasts. The Version 2.4.0, Swofford 1986) using the Branch and Bound op- tion. A consensus cladogram was constructed from the most par- position of the chloroplast in Didymosphenia appar- simonious cladograms using the subroutine Contree. Putative ently has not been reported. Our unpublished ob- synapomorphies (shared, derived characters) were mapped on the servations on D. geminata (Lyngb.) M. Schm. indicate consensus cladogram and identified numerically. the chloroplast is dorsal, and this orientation is in- dicated by Meyer’s (1929) illustration of a dorsally- RESULTS placed pyrenoid during reduction division in this species. The same rationale described for [ 171 and The consensus cladogram (Fig. 2) indicates the [18] above can be applied here to consider both lineage of freshwater APF-bearing cymbelloid and ventrally rotated (a) and dorsally rotated (b) chlo- gomphonemoid diatoms and their close allies is de- roplasts as derived and unordered. fined by the presence of APF’s, the IAlaa type of 20. Dzfference between dorsal and ventral mantle sexuality described by Geitler, as well as a dorsal heights. Krammer (198 1) indicates that species of stigma, distal raphe ends that are deflected ventrally, Cymbella s. 1. possess a difference in valve mantle and a ventrally rotated chloroplast with ventral py- heights. Of the terminal taxa considered here only renoid and dorsal nucleus. The lineage is divided Cymbella, C. mexicana and Encyonema possess this fea- into two clades; one comprised predominately of ture, which, owing to its absence in the outgroups, cymbelloid diatoms, the other comprised almost en- is considered derived. tirely of gomphonemoid diatoms. The cymbelloid 2 1. Apical porejields present. All terminal taxa, ex- clade is defined by asymmetry about the apical axis cept Encyonema, possess apical pore fields. This fea- and contains the taxa Reimeria, Encyonema, Cymbella, ture is lacking in the outgroups, suggesting presence C. mexicana and the gomphonemoid genus Didymo- of APF’s is derived. sphenia. The gomphonemoid lineage is defined by 382 JOHN P. KOCIOLEK AND EUGENE F. STOERMER the presence of slit-like stigmata and contains Bre- division and gamete pairing as well as an orientation bissonia, Gomphocymbella, Gomphonema and the two of cytological and valve features similar to Encyo- subgroups of Gomphoneis. nema, Reimeria and Gomphonema, was ancestral to the Within the cymbelloid lineage, Reimeria branches entire lineage. That this ancestral form resembled first and possesses the autapomorphic (derived, but a member of Placoneis is strengthened by the obser- unique) feature of unequal APF’s. The rest of the vation of Cox (1987) which shows recurved internal lineage is defined by having dorsal and ventral man- proximal raphe ends, a feature also observed in many tles of different heights. Encyonema branches next gomphonemoid and cymbelloid species (Dawson and, according to the hypothesis, members lack 1973c, Krammer 1982b, Kociolek and Stoermer APF’s through secondary loss. The remaining mem- 1986b, c). The cymbelloid diatoms and gompho- bers of the cymbelloid clade (Cymbella, C. mexicana nemoid diatoms then each diverged independently and Didymosphenia) form a group which is defined from the naviculoid archetype. by the features of differentiated APF’s, lack of an Within the cymbelloid line, Reimeria, then Ency- intermissio, APF’s not bisected by the raphe, ven- onema, branched, and the eventual evolution of Cym- trally placed stigmata, distal raphe ends deflected bella s. s. and its allies was accomplished through a dorsally, and a dorsally rotated chloroplast with a polarity reversal of organellar and valve features, as dorsal pyrenoid and ventral nucleus. The genus Cym- hypothesized by Mann (1983b). It seems likely that bella branches at this point and is defined by the members of Cymbopleura, a heterogenous group of presence of dendritic stigmata. Didymosphenia and cymbelloid diatoms, may have branched indepen- C. mexicana are shown to share a more recent com- dently at various points along the cymbelloid clade. mon ancestor based on the features of complex fo- Additional observations on the ultrastructure and raminae and conical, silica-covered stigmata. The cytology of these enigmatic taxa will be necessary to genus Didymosphenia is shown to have the derived assess their systematic positions. features of asymmetry about the transapical and per- The analytical result which shows Didymosphenia valvar axes, an APF at one pole only, and to have and Cymbella mexicana to share a more recent com- secondarily lost differences in valve mantle height. mon ancestor than either does with Cymbella s. s. or In the lineage predominated by gomphonemoid other gomphonemoid diatoms suggests that the fea- taxa, the genus Brebissonia branches first, and it is ture of valve symmetry, which has been given so without any defining features. Asymmetry about the much weight in the construction of classification transapical and pervalvar axes, APF’s restricted to schemes for diatoms, may not be a good indicator one pole and differentiated APF’s define the rest of of genealogical relationships, particularly in this the clade. Asymmetry features and APF’s at one pole group. Cox (1979) has previously made this point are considered convergences between this gompho- with regard to Gyrosigma Hass. and Pleurosigma W. nemoid lineage and Didymosphenia. At this level of Sm. A consequence of the specific result here is that hierarchy in the gomphonemoid line there is an un- for the genus Cymbella s. s. to be a monophyletic resolved trichotomy between Gomphocymbella, Gom- group (sensu Hennig 1966) either Didymosphenia must phonema and Gomphoneis. Asymmetry about the api- be included in the genus or be joined by C. mexicana cal axis (a convergence with the cymbelloid lineage) outside Cymbella. In either case the results clearly defines Gomphocymbella whereas presence of septa, indicate Didymosphenia to be more closely related to pseudosepta and marginal laminae define the genus Cymbella species than to other gomphonemoid dia- Gomphoneis. Gomphonema lacks defining features. toms. Within Gomphoneis, G. elegans is defined by undif- Within the gomphonemoid line, the two subgroups ferentiated APF’s and lack of stigmata (both fea- of Gomphoneis are shown to be joined by the features tures are considered evolutionary reversals) and the of marginal laminae, septa and pseudosepta. The G. herculeana subgroup is defined by the presence features of marginal laminae and multi-punctate of an axial plate. striae were identified by Krammer and Lange-Ber- talot (1985) to distinguish Gomphoneis. Expression of DISCUSSION doubly-punctate striae in species of cymbelloid dia- Previously published interpretations of interre- toms (Hustedt 1942, 1944, Okuno 1974, Germain lationships of the taxa included among the fresh- 198l), Reimeria (Krammer 1982a, Kociolek and water APF-bearing cymbelloid and gomphonemoid Stoermer 1987), Gomphonema (Hustedt 1942, 1945, diatoms and their close allies, which envision cym- Krammer and Lange-Bertalot 1985) and Brebissonia belloid diatoms as primitive, gomphonemoid dia- (Mahoney and Reimer 1986) suggest this feature toms as derived, and these two groups joined by may actually be derived for the whole group and intermediates such as gomphocymbelloid species thus not a defining feature of Gomphoneis. By the (Cleve 1894) or Reimeria sinuata (Krammer and criterion of presence of marginal laminae, other Lange-Bertalot 1985)) are not supported by the gomphonemoid diatoms which possess this structure present results. Results of the cladistic analysis sug- (e.g. Gomphopleura Reich., R. Mahoney, pers. comm.) gest that a Placoneis-like taxon, having undifferen- would have to be included in the genus. The diatom tiated APF’s at both poles, a process of reduction Gomphonema ventricosum Greg. has been reported to PHYLOGENETIC RELATIONSHIPS OF DIATOMS 383

possess a septum (Dawson 1973c) and may represent and APF’s restricted to the footpole may be the a form intermediate between Gomphonema and Gom- result of a single process. That these three features phoneis. also appear to be 100% correlated, even in appar- Although not included as terminal taxa in the ently unrelated groups such as the araphid genus present investigation, comment here on the taxo- Licmophora C. A. Ag. (Hendey 1964, Wahrer et al. nomic position of some doubly-punctate species of 1985), suggests that a single ontogenetic pathway Gomphonema (G. olivaceum (Lyngb.) Kiitz., G. quad- might produce gomphonemoid heteropolarity. ripunctatum Ostr.) seems appropriate. Dawson (1974) The cladistic analysis of phylogenetic relation- included these species in Gomphoneis on the basis of ships among the freshwater APF-bearing cymbel- their dissimilarity to most other Gomphonema species, loid and gomphonemoid diatoms and their close al- including the presence of doubly-punctate striae. lies represents a testable hypothesis concerning Kociolek and Rosen (1984) argued for their reten- interrelationships of the terminal taxa and the evo- tion in Gomphonema due to a lack of an axial plate. lution of characters. As our understanding of the Lack of marginal laminae in these species was used pool of taxa presently classified in Navicula becomes as evidence to retain them in Gomphonema (Krammer better known and as the ultrastructure of other cym- and Lange-Bertalot 1985, 1986). Undifferentiated belloid and gomphonemoid species is described, the APF’s and septa have been observed in these species validity of this hypothesis as an estimator of the (Dawson 1974, Okuno 1974, see also Skabitschevsky groups’ genealogy may be evaluated. 1984). Additionally, because these species may be astigmate (like Gomphoneis transsilvanica (Pant.) We thank J. R. Johansen, J. C. Kingston, R. L. Lowe, E. C. Theriot Krammer in Krammer and Lange-Bertalot) or have and D. M. Williams, and an anonymous reviewer, for providing several stigmoids about the central area (as in G. helpful comments on earlier versions of this manuscript. This elegans), an argument can be made for their affinity research was funded by Grants BSR-8500263 and BSR-8722516 of the National Science Foundation to EFS. Contribution No. with th.e G. elegans subgroup of Gomphoneis. The 504 of the Great Lakes Research Division. condition of marginal laminae would have to have been secondarily lost. Following the generic defi- Anonymous. 1703. Two letters from a gentleman in the country, nition proposed by Krammer and Lange-Bertalot relating to Mr. Leeuwenhoek’s letter in Transaction No. (1985), the taxonomic implication would be that the 283. Philos. Trans. (Land.). 288:1494-501. G. olivaceum/G. quadripunctatum group best belongs Cholnoky, B. J. 1929. Beitrage zur Kenntnis der Auxosporen- in Gomphoneis. Thus, Gomphoneis is a polythetic tax- bildung. Arch. Protistenk. 68:471-502. Cleve, P. T. 1894. Synopsis of the naviculoid diatoms. Part 1. on, that is not all taxa exhibit the features which K. Svenska Vetens-Akad. Handl. 26:l-194. define it (Wiley 1981). Similarly, the entire ingroup Cox, E. J. 1977. Variation in valve structure between species of considered in the present report is polythetic, since the diatom genus Cymbella C.A. Ag. Nova Hedwigaa 28:427- although presence of APF’s help to define the lin- 49. - 1979. Symmetry and valve structure in naviculoid dia- eage, Encyonema species lack them yet are still in- toms. Nova Hedwigia Beih. 64: 193-206. cluded in the clade. 1987. Placoneis Mereschkowsky: The re-evaluation of a The genus Gomphonema, as shown in the cladistic diatom genus originally characterized by its chloroplast type. analysis, lacks any defining characters, i.e. it is a Diatom Res. 2:145-57. Dawson, P. A. 1972. Observations on the structure of some “Not-A” group (Eldredge and Cracraft 1980). In forms of Gomphonema parvulum Kiitz. I. Morphology based many cases “Not-A” groups represent heterogenous on light microscopy, and transmission and scanning electron assemblages of taxa, sometimes sharing only the microscopy. Br. Phycol. J. 7:255-71. commonality of lacking derived features (Wiley -1973a. The morphology of the siliceous components of 1981). The myriad of forms assigned to Gomphonema Didymosphenia geminata (Lyngb.) M. Schm. Br. Phycd. J. 8: 65-78. (Dawson 1973c, Krammer and Lange-Bertalot 1986, -1973b. Further observations on the genus Didymosfihenia Kociolek et al. 1988) suggest this possibility. Addi- M. Schmidt-D. sibirica (Grun.) M. Schm. Br. Phycol. J. 8:197- tional studies on the ultrastructure of Gomphonema 201. species are warranted to identify whether they com- -1973c. Observations on some species of the diatom genus Gomphonema C.A. Agardh. Br. Phycol. J. 8:413-23. prise a monophyletic taxon. 1974. Observations on diatom species transferred from With regard to character evolution, Mann (1 983b) Gomphonema C.A. Agardh to Gomphoneis Cleve. Br. Phycol. J. suggested the features of ventrally-placed stigmata, 9:75-82. external distal raphe ends that are deflected dor- Desikachary, T. V. 1957. Electron microscope studies on dia- sally, and a dorsally-rotated chloroplast possibly arose toms. J. R. Micr. Sac. 76:9-36. Eldredge, N. & Cracraft, J. 1980. Phylogenetic Patterns and the together via polarity reversal from the opposite, an- Evolutionary Process. Columbia University Press, New York, cestral condition, and his hypothesis is supported by 349 pp. our analysis. That these features were derived from Fricke, F. 1902. Tafeln 233-238. In Schmidt, A. [Ed.] 1874- a single process is suggested by their 100% corre- 1959, Atlas der DiatomaceenKunde. R. Reisland, Leipzig, pls. 1-418. lation (see Mayr 1969). Similarly, the three features Geitler, L. 1958. Notizen uber Rassenbildung, Fortpflanzung, of valve asymmetry in gomphonemoid diatoms, Formweschel und m,orphologie Eigentiimlichkeiten bei pen- namely asymmetry with regard to the transapical naten Diatomeen. Osterr. Bot. 2. 105:408-42. axis, asymmetry with regard to the pervalvar axis, - 1973. Auxosporenbildung und Systematik bei pennaten 384 JOHN P. KOCIOLEK AND EUGENE F. STOERMER

Diatomeen und die Cytologie von Cocconeis-Sippen. &err. cillariophyceae). IV. Valve ultrastructure and systematic po- Bot. 2. 122~299-321. sition of Gomphoneis elegans. Trans. Am. Microsc. SOC.In press. - 198 1. Die Lage des Chromatophors in Beziehung zur Kociolek,J. P., Stoermer, E. F. & Bahls, L. L. 1986. Observations Systematik von Cymbella-Arten (Bacillariophyceae). PI. Syst. on North American Gomphoneis (Bacillariophyceae). 111. Gom- Evd. 138:153-6. phoneis septa (Mogh.) comb. nov. Can. J. Bot. 64:2764-8. Gerloff, J. & Helmcke, J-G. 1977. Tafel 987. In Helmcke, J-G., Kociolek, J. P., Yang, J-R. & Stoermer, E. F. 1988. , Krieger, W. & Gerloff, J. [Eds.] Diatomeenschalen im Elektro- ultrastructure and systematic position of the Gomphonema nenmikroskopischen Bild. Teil X.J. Cramer, Vaduz. grovei-speciescomplex. Nova Hedwigza. In press. Germain, H. 198 1. Flore des Diatomies. Diatomophycies Eaux Douces Krammer, K. 1979. Zur Morphologie der Raphe bie der Gattung et Saumitres du MassifArmoricain et des Contries Voisires d’Eu- Cymbella. Nova Hedwigia 21:993-1029.

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J. Phycol. 24, 385-393 (1988)

ULTRASTRUCTURE OF FERTILIZATION IN A CRYPTOMONAD’

Paul Kugrens2 Department of Biology, Colorado State University, Fort Collins, Colorado 80523 and Robert E. Lee Department of Anatomy, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT do not fuse. Meiosis presumably is zygotic, but the stages By means of various electron microscopic techniques, the of post-karyogamy remain to be elucidated. ultrastructure of fusing gametes in a cryptomonad is de- Key index words: cryptomonads; fertilization; ultra- scribed for the first time. The isolate used in this study is structure bisexual, and vegetative cells may act as isogametes. Plas- mogamy usually is initiated at the posterior end of one gamete and the mid-ventral region of the other gamete. A The most common means of reproduction in cryp- posterior, pointed protuberance may be a specialized mat- tomonads is by mitotic cell division (Oakley and ing structure which initiates the fusion process. Fusion Dodge 19’76, Oakley and Bisalputra 1977, Mc- proceeds toward the anterior end, forming a quadrzfa- Kerracher and Gibbs 1982, Oakley and Santore gellate cell which becomes spherical and settles to the bottom 1982, Meyer and Pienaar 1984a, b). Sexual repro- of the culture jask. The quadrijiagellate, spherical cell duction apparently is rare and generally is perceived contains tuio nuclear--chloroplast complexes to be absent because direct evidence for a sexual which remain intact throughout karyogamy. During kary- process is lacking. For instance, Wawrik provided ogamy the nuclei are positioned close to each other and brief descriptions and diagrams of gamete fusion in become lobed on the sides where fusion takes place. At the field collections of (Wawrik 1969) and points where the lobes touch, the nuclear membranes break (Wawrik 197 l), but unfortunately these down and direct karyogamy is initiated. Nuclear fusion are not documented with photomicrographic evi- continues and eventually a single zygotic nucleus isformed. dence. Furthermore, the diagrams do not depict The zygote nucleus and the two and chlo- karyogamy. Hill and Wetherbee (1986), by utilizing roplasts become enclosed in a common periplastidial com- fluorescence microscopic techniques on the marine partment. The nucleomorphs, however, remain apart and cryptomonoad Proteomonas Hill and Wetherbee, de- tected haploid and diploid cells in a clonal culture. In addition, electron microscopy of Proteomonas cells Accepted: 9 May 1988. provided structural differences between the two cell * Address for reprints. types, suggesting a diplomorphic history. The