Morphological Variation in Diploneis smithii and D. fusca (Bacillariophyceae)

Arch. Protistenkd. 144 (1994): 249-270

ARCHIV FUR

© by Gustav Fischer Verlag Jena

PROTISTEN KUNDE

Morphological Variation in Diploneis smithii and D. fusca (Bacillariophyceae) STEPHEN

J. M.

DROOP

Royal Botanic Garden Edinburgh, Edinburgh, UK Summary: Morphological variation within the Diploneis smith;; - D. fusca complex has been examined critically in a single collection from Oban, Scotland. Eleven distinct sympatric morphotypes can be identified. Each exhibits a recognizable size-reduction sequence in which all critical characters are stable. Measurements of dimensions, striation density and rectangularity are combined with two other characters (shape of central raphe endings and the arrangement of pores on the surface of the valves) in statistical analyses (including Principal Components Analysis and Hierarchical Clustering Analysis) that demonstrate objectively the validity of the groupings. The implications of such discontinuities are discussed with respect to current knowledge of patterns of reproduction in diatoms, breeding barriers between similar races, and phenotypic plasticity. The morphotypes probably represent taxa with different ecological preferences, and should in due course be given taxonomic recognition to avoid loss of information. Distinct morphotypes similar to those in D. smithii - D. fusca occur within the species of other genera, and the phenomenon may be very widespread among diatoms.

Key Words: Diatoms; Morphological variation; Diploneis smith;; (BREBISSON) CLEVE; Diploneis fusca (GREGORY) CLEVE; Breeding strategies.

Introduction Diploneis smithii (BREBISSON) CLEVE was first described as Navicula elliptica by W. SMITH (1853, p. 48), using material from Poole Bay, collected in September 1850. Later, SMITH 1856, p. 92), gave it the name Navicula smithii (at de BREBISSON'S suggestion), since KUTZING (1844) had already used the name N. elliptica for a freshwater species. Since then, a large number of varieties or related species have been described: VANLANDINGHAM'S (1969) entry for D. smithii runs to over three pages, with 26 synonyms listed under the species, followed by a large number of accepted and excluded infraspecific taxa together with their synonyms. The taxonomy af D. fusca (GREGORY) CLEVE is perhaps even more complex than that of D. smithii, since the species as commonly delimited should be called D. aestiva (DONKIN) CLEVE, "aestiva" being the earliest

epithet to have been used for it at species level within Diploneis or Navicula (DONKIN 1858); "fusca" was described first only as var. B of N. smithii (GREGORY 1857) and so cannot claim priority. VANLANDINGHAM (1969) gives D. aestiva as a synonym of D. fusca var. aestiva; his entry for D. fusca runs to more than two pages, with 21 synonyms under the species, followed again by a large number of accepted and excluded taxa and their synonyms. Both species, clearly, are complex and variable, but most authors agree that there is a single diagnostic character separating them (Table 1): the microstructure of the striae. In D. fusca the transapical ribs are crossed by several longitudinal ribs which apparently divide the striae into discrete chambers; in D. smithii, the striae are undivided and consist instead of double rows of alternating pores (compare Figs. 19 and 33).

250

S. J. M. DROOP

Table 1. Diagnostic separation of D. smithii and D. fusca in the keys of some published texts. Ellipsis (...) indicates further (irrelevant) dichotomies not shown. Author, date, page

Dichotomy

Text

CLEVE (1894), p. 78

25

Costae crossed by several longitudinal ribs ... D·fusca Costae alternating with double rows of alveoli ... D. smithii

CLEVE-EuLER (1953), p. 66

D36++

Uingsrippen deutlich: D·fusca Doppelreihen von Punkten deutlich: D. smithii

HUSTEDT (1927-66), Teil2, p. 582, 583

BIb

Transapikalrippen nicht von Uingslinien gekreuzt. AuBenwande deutlich poroid: IV Gruppe (which contains D. smithii)

Ibid, p. 583

BII b2

Transapikalrippen von Uingslinien (scheinbaren oder echten Rippen) gekreuzt . . . Schalen ohne echte Langsrippen, nur die AuBenwande der Kammem stark poroid, Kammem daher im Innem ungeteilt: VII Gruppe (which contains D. fusca)

This character alone might indeed be enough to distinguish the species were it not for the occurrence of intermediate forms that exibit ''fusca'' structure near the raphe and "smithii" structure closer to the valve margins (e.g. Fig. 2). HUSTEDT (1927-66) discussed the taxonomic implications of this phenomenon, and considered that all forms should be classified within the same variable species; he declined to put this into effect and make the necessary new combinations, however, because of the enormous nomenclatural disruption it would cause. He excluded some other species from the complex, although his arguments seem rather tenuous: D. vacillans (A. S.) CLEVE, although acknowledged to belong to the "Formenkreis" of D. smithii, has weak longitudinal lines crossing the ribs that divide the striae into rectangular chambers each of which contains a pair of adjacent pores. D. graeffii (GRUN.) CLEVE was excluded from the D. smithii-fusca complex (although HUSTEDT acknowledged a similarity with D. fusca) on the grounds that the longitudinal lines crossing the ribs completely divide the striae into chambers (whereas in

D. fusca the longitudinal lines are shallower than the transapical ribs and only seem to subdivide the striae). D. littoralis (DONKIN) CLEVE and D. parca (A. S.) BOYER were not included as part of the complex on account of their distinctive appearance, with rather narrow parallel longitudinal canals. One sample in the collections of the Royal Botanic Garden Edinburgh (E 1014) was found to be very rich in species of Diploneis. Many valves were found belonging to the D. smithii-D. fusca complex, i. e. oblong or oval valves with pores or ribs clearly visible between the main transapical ribs so including those with a superficial resemblance to D. vacillans and D. graeffii, but excluding D. littoralis and D. parca which are easily identifiable by their narrow and parallel longitudinal canals. D. graeffii can be excluded from the discussion since none of the valves examined had striae completely divided into chambers (the longitudinal ribs were all shallower than the transapical ribs - see, for example, Figs. 48,49). In addition, HUSTEDT (1927-66) recorded its distribution as "aus warmeren Meeren". I have not been able to confirm HARTLEY'S (1986) British record for it. The valves in E 1014 exhibited a wide range of variation covering ''Jusca'', "smithii" and "vacillans" types, and so a detailed analysis was undertaken to determine whether there is a continuum of variation or a series of well-defined, though similar, forms. The starting point (null hypothesis) for the investigation was HUSTEDT'S viewpoint (1927-66) that most marine oval or oblong Diploneis with pores or chambers clearly visible in the striae should be classified in the same species. This implies that the variation encountered in a population can be ascribed to phenotypic plasticity or has little or no taxonomic significance; that if subgroups (usually varieties) can be distinguished, there is sufficient overlap between them to preclude complete separation. Whether or not this viewpoint stands up to close scrutiny when applied to sample E 1014 could have far-reaching implications, not only for the systematics of D. smithii sensu lato, but also for diatom systematics as a whole.

Materials and Methods The material was collected in Feb. 1991 from a sandy beach near Oban, U. K., from close to the low-tide mark (Grid Ref. NM 862 328), and is housed at the Royal Botanic Garden Edinburgh as sample E 1014. Diatoms were collected using an adaptation ofthe EATON & Moss (1966) lens-tissue method, acid-cleaned, and mounted in Naphrax on glass slides. Photographs were taken on Kodak Technical Pan using a Reichert Polyvar photomicroscope and a Zeiss DSM 962 scanning electron microscope.

Morphological Variation in Diploneis smithii and D. fusca

Measurements were made on a Zeiss Axiophot photomicroscope fitted with a CCTV camera connected to image-analysis equipment: a 486-chip PC-compatible microcomputer with a frame-grabber, running the imageanalysis program OPTIMAS (BioScan Inc.). Broken diatoms, and those lying squint (more than about 1 flm between the vertical focus of opposite sides or ends) were rejected (about 10% of the total). Measurements of dimensions and shape were taken from the outline of the diatom identified semi-automatically: the computer was programmed using OPTIMAS to find the outline of each diatom by identifying the boundaries between dark and light areas, and ignoring boundaries below a certain length and those fully enclosed within others. The process was automated using a macro facility requiring minimal operator input. The length of the diatom was taken as the distance between the two points on the outline that were furthest apart; the width as the sum of the perpendicular distances from the long axis to the most distant point on each side. Measurement of striation densities was achieved by instructing the computer to recognize the darkest points along a line defined by the user. The distances from the end of the line to each of these points could then be used to calculate the number of striae in 10 flm along the line, usually averaged from two lines (Fig. Ib). It was felt that the study would be more credible if, in addition to qualitative analysis, objectivity could be ensured through a numerical phenetic approach. The following statistical methods were chosen: A Principal Component Analysis (PCA) was performed on the data matrix using Minitab statistical software (Minitab Inc., 3081 Enterprise Drive, State College, PA 16801, USA); the data were not transformed, and the analysis was performed on the correlation matrix. PCA rearranges the variation in a sample into a number of orthogonal and dimensionless variates (the Principal Components, PCs) in such a way that most of the variation (in as many variates as have been scored) is expressed by the first few PCs, which can easily be visualized with only very few plots. The advantage of PCA in this context over, for example, discriminant analysis is that it treats all specimens alike, with no predefined groupings used to bias the results. PCA has the advantage that it allows the overall pattern of variation to be summarized and displayed, but it does not analyse relationships between the individual objects studied (in this case diatom valves), so a more rigorous approach was also adopted. A Hierarchical Clustering Analysis (HCA) was performed on the data to measure the similarity between each pair of valves, using the numerical taxonomy program NTSYS (Exeter Software, 100 North Country Road, Building B, Setauket, NY 11733, USA). All characters were treated as ordered continuous or multistate, and there were no missing values. The dissimilarity matrix was calculated using average taxonomic distances defined _I ~ 2 by: dij ='1 (lIn ":'k(Xk.i + xkj ) ) in which i and} are operational taxonomic units (OTUs, in this case valves), k is a character, x is a value or state, and n is the number of characters. Clusters were formed using

251

Unweighted Pair-Group Method, Arithmetic Average (UPGMA) (SNEATH & SOKAL 1973). The dissimilarity matrix was compared with the cophenetic value matrix calculated from the dendrogram. The graphs were created using SigmaPlot (Jande! Scientific. P.O.Box 7005, San Rafael, CA 94912-8920, USA).

Fig. 1. Characters used in qualitative and morphometric analyses: (a) maximum length and width along and perpendicular to main axis of diatom; (b) striation density note the position of measurement, close to longitudinal canal, in diagonally opposite quarters of the valve; (c) shape of central raphe ending (CRE) - not in focus in this photograph: hooked in most forms but straight and rounded in this morphotype (see Figs. 39-49 for better illustrations); (d) 'fusca"-structure - striae divided into chambers; (e) "smithii"-structure - pores in alternating pairs; (f) outline of diatom for measurement of rectangularity - more oblong diatoms have a larger area for a given length and breadth, and so a higher rectangularity; (g) longitudinal canal; (h) central nodule; (i) raphe sternum furrow.

252

S. J. M. DROOP

Characters investigated Nine morphological characters were examined and these are illustrated and explained in Fig. I. All of them, except those involving the longitudinal canals, were used in the statistical analyses. Dimensions (11m) (Fig. la): Variation in length and width is an inevitable consequence of the life-cycle of most diatoms (see WIEDLING 1948 and GEITLER 1932 for some exceptions). As a general rule, though, this variation occurs only within a restricted range that is more or less fixed for each genotype (GEITLER 1932), and so can yield valuable taxonomic information. Measurements were made using image-analysis and were accurate to within about half a micron. Striation density (no.110 11m) (Fig. Ib): The periodicity of pattern on diatom valves has long been used as a taxonomic character, and is still one of the most important. Whereas in the past diatomists have had to make do with measuring the periodicity of striae to a precision of 10% or worse, it is now routinely possible to measure striation densities to a precision of at least as good as 2%, even without imageanalysis (DROOP 1993 and in prep.); here measurements were made using image-analysis (see above). Shape of external central raphe endings (Figs. Ic, 39-56): This character has apparently not been used before in Diploneis, but proved important in confirming groupings suggested by other characters such as valve outline and microstructure. Figs. 50-53 show part of the same valve at different focus levels; it is quite clear that the roundness of the raphe endings is not a focusing artefact and can be assessed reliably using the light microscope. The scanning micrographs (Figs. 54-56) reinforce the distinction. For the statistical analyses, 0 = bilaterally symmetrical, round; I =slightly asymmetrical but not hooked; 2 =clearly hooked to one side of the valve (in all valves seen that exhibit this character state, both ends were hooked to the same side of the valve). Microstructure ofvalve surface (Figs. Id, e, 39-49): This is the character that has caused most of the confusion within the complex. Valves with "smithii"-structure have alternating pores along the striae with no longitudinal ribs (Fig. Ie); those with 'jusca"-structure have longitudinal ribs that seem to divide the striae into more or less square chambers (Fig. Id); those with "vacillans"-structure have adjacent pairs of pores separated from the next pair transapically by thin longitudinal ribs, usually closer together than the main transapical ribs (e. g. Fig. 47). Most valves have more than one of these structure-types, usually with ''fusca''- or "vacillans"-structure close to the central axis, and "smithii"-type structure close to the margins. The extent of each type of structure in a valve was scored to test the character's taxonomic significance in this context. For the purposes of the statistical analyses, ''fusca''- and "vacillans"-structures were grouped together: each valve was scored for the ratio of ''fuscalvacillans'': "smithii"structure over the whole valve surface excluding the longi-

tudinal canals and the area enclosed within them: thus Fig. I shows ''fusca''-structure over approximately onethird. Scores ranged from 0 to 10 in intervals of 0.5. Shape of valves (Fig. If): Differences in diatom shapes can easily be detected by eye, but taxonomic assessment of shape is difficult, especially where differences are slight and where objectivity is required. For the statistical analyses, the character was simplified to a measure of rectangularity: the area of the diatom expressed as a proportion of the area of its enclosing rectangle (Fig. If). (See DROOP in prep. for a fuller discussion of the use of rectangularity as a measure of shape.) Shape oflongitudinal canals (Fig. Ig-i): This can be split into at least three characters. They are somewhat subjective, and it has not been possible to quantify them adequately for statistical analyses; they are included here as supporting evidence. a) Shape enclosed by abaxial margins of both canals: lensiform (e.g. Fig. 6), or more or less rhombic (e.g. Fig. 3); b) Width of each longitudinal canal at centre relative to width of central nodule (Fig. Ih): longitudinal canal wider than central nodule (e. g. Fig. 6), or longitudinal canal about the same width as or narrower than central nodule (e. g. Fig. 33); c) Shape of raphe sternum furrow (Fig. Ii): furrows clearly constricted some way short of their central ends (e. g. Fig. 38); furrows not or only slightly constricted close to central nodule (e. g. Fig. 6).

Observations and Results A total of 535 valves were measured from three slides from sample E1014. This represents all ofthe valves belonging to the D. smithii complex on the three slides, except for one particularly distinctive and cornmon form (Morphotype 7, Figs. 21-24), where measurements were limited to 100 valves to save time; in this case, the valves were selected randomly (the first 100 valves encountered during systematic scanning of a slide). Rather than showing the variation to be continuous, a preliminary qualitative analysis revealed eleven groups, clearly identifiable and represented by enough valves to get a reasonable idea of the range of sizes within a size-reduction sequence. The term "morphotype" will be used for these groups; in the terminology of GILMOUR & HESLOP-HARRISON (1954), they would be "phenodemes". Figs. 2-30 and 33-38 illustrate the eleven morphotypes, all at the same scale. Each is represented by three or four individuals of different sizes to show the range of aspect ratios in each, and to stress the similarity between the valves of each. In addition, there were two individual valves that did not fit any of the other morphotypes (Figs. 31, 32, to the same scale as Figs. 2-30, 33-38). Figs. 39-56 are close-up views of valves (LM and SEM) to show details of microstructure and raphe.

Morphological Variation in Diploneis smithii and D.fusca

Figs. 2-7. Morphotypes 1-2. Scale bar =20 !Jm. Figs. 2-4: Morphotype 1; Figs. 5-7: Morphotype 2.

253

254

S. J. M.

DROOP

11

12

Figs. 8-14. Morphotypes 3-4. Scale bar =20 /lm. Figs. 8-10: Morphotype 3; Figs. 11-14: Morphotype 4.

13

Morphological Variation in Diploneis smithii and D. fusca

Figs. 15-20. Morphotypes 5-6. Scale bar = 20 fJill. Figs. 15-17: Morphotype 5; Figs. 18-20: Morphotype 6.

255

256

S. J. M. DROOP

Morphological Variation in Diploneis smithii and D. fusca

257

35

38

Figs. 33-38. Morphotypes 10-11. Scale bar = 20 f.lm. Figs. 33-35: Morphotype 10; Figs. 36-38: Morphotype II. Figs. 21-32. Morphotypes 7-9, and unmatched valves A and B. Scale bar = 20 f.lm. Figs. 21-24: Morphotype 7; Figs. 25-27: Morphotype 8; Figs. 28-30: Morphotype 9; Fig. 31: Unmatched valve A; Fig. 32: Unmatched valve B.

258

S. J. M. DROOP

,-

...

•

"

~

• ,

,",,-

~

,~

."

~ ~

39 ) '.

w..--i ~

II..;

,~'

:-r;

'III

• '1

= 'j : :

.- ft

40

.... .... '

~

~

~

,,,'

~

....

42 ~C'2

Figs, 39-56, Details of central raphe endings and valve structure. For Figs. 39-53, scale bar (in Fig. 45) = 10 11m; Figs. 54-56 scaled individually: scale bars =211m. Figs. 39-49: Morphotypes I-II: Fig. 39: Morphotype 1; Fig. 40: Morphotype 2; Fig. 41: Morphotype 3; Fig. 42: Morphotype 4; Fig. 43: Morphotype 5; Fig. 44: Morphotype 6; Fig. 45: Morphotype 7; Fig. 46: Morphotype 8; Fig. 47: Morphotype 9; Fig. 48: Morphotype 10; Fig. 49: Morphotype 11. Figs. 50-53: Detail of one valve of Morphotype 10 at different foci to demonstrate that rounded central raphe endings are not a focusing artefact and can be assessed using the light microscope. Figs. 54-56: SEM micrographs of central raphe endings: Fig. 54: Morphotype 2; Fig. 55: Morphotype 10; Fig. 56: Morphotype II.

Morphological Variation in Diploneis smithii and D. fusCG

Descriptions of morphotypes

259

The following descriptions complement Table 2 and Figs. 57-61.

central nodule. Raphe-sternum furrow parallel-sided, not noticeably constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Fig. 41).

Morphotype 1 (Figs. 2-4, 39; 98 valves measured): Medium- to large-sized coarsely-striate, oblong-elliptical diatoms. "fusca" -structure occupying about onethird of the valve surface distal to the longitudinal canals (Fig. 39). Longitudinal canals and raphe system forming a narrowly rhombic shape. Longitudinal canals wider than central nodule. Raphe-sternum furrow slightly convex, not noticeably constricted close to the central nodule. Central external raphe endings round, bilaterally symmetrical (Fig. 39).

Morphotype 4 (Figs. 11-14,42; 71 valves measured): Medium-sized finely-striate, oblong-elliptical diatoms. "vacillans" -structure occupying about three-quarters of the valve surface distal to the longitudinal canals (Fig. 42). Longitudinal canals and raphe system forming a lensiform shape. Longitudinal canals wider than central nodule. Raphe-sternum furrow parallel-sided, not noticeably constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Fig. 42).

Morphotype 2 (Figs. 5-7, 40, 54; 66 valves measured): Medium- to large-sized coarsely-striate, elliptical diatoms. ''fusca'' -structure occupying about one-half of the valve surface distal to the longitudinal canals (Fig. 40). Longitudinal canals and raphe system forming a lensiform shape. Longitudinal canals wider than central nodule. Raphe-sternum furrow parallel-sided, not noticeably constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Figs. 40, 54).

Morphotype 5 (Figs. 15-17,43; 17 valves measured): Medium- to large-sized finely-striate, oblong diatoms. "vacillans"-structure occupying about one-quarter of the valve surface distal to the longitudinal canals (Fig. 43). Longitudinal canals and raphe system forming a narrowly rhombic shape. Longitudinal canals narrower than central nodule. Raphe-sternum furrow slightly convex, slightly constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Fig. 43).

Morphotype 3 (Figs. 8-10,41; 44 valves measured): Medium- to large-sized finely-striate, elliptical diatoms. "vacillans" -structure occupying about three-quarters of the valve surface distal to the longitudinal canals (Fig. 41). Longitudinal canals and raphe system forming a !ensiform shape. Longitudinal canals wider than

Morphotype 6 (Figs. 18-20, 44; 8 valves measured): Medium- to large-sized coarsely-striate, elliptical diatoms. ''fusca'' -structure occupying about one-tenth of the valve surface distal to the longitudinal canals (Fig. 44). Longitudinal canals and raphe system forming a narrowly rhombic to almost lensiform shape. Longi-

57

45 40

-a -... :t

.1:1

•'tI

• Morphotype 1 ... Morphotype 2 • Morphotype 3 A Morphotype 4 • Morphotype 5 o Morphotype 6 '" Morphotype 7 o Morphotype 8 £> Morphotype 9 o Morphotype 10 o Morphotype 11

35 30 25 20

• Valve A ... Valve B

15 10 20

30

40

50

60

70

80

90

100

Length (~m)

Fig. 57. Plot of width against length (characters I and 2 in Table 2) for all 535 valves scored. Envelopes enclose outliers of each morphotype.

Table 2. Characters and character-states for the morphotypes and individual valves scored. Columns are characters; row blocks are morphotypes (col. M = morphotype number); col. n. = sample size; J.1 = mean; (J = sample standard deviation. Character 3 is Striation density. In Character 4, CRE is Central Raphe Endings; 0 = rounded, 1 = slightly asymmetrical, 2 = hooked. In Character 5, f = fusca-structure, v = vacillans-structure, s = smithii-structure; round brackets indicate secondary state; 0 = all smithii-structure, 10 = all fusca- or vacillans-structure. Char.:

M

3

5 Valve struct.

6 Valve shape I rectangularity

7(a) Shape enclosed by longit. canal

7(b) Width longit. canal cf central nodule

7(c) Shape of raphe sternum furrow

(f)/s [1.5-5] J.1 = 3.11 (J = 0.76

Oblong-elliptical

Rhombic

Wider

Slightly convex

f/s [4-6] J.1 = 5.03 (J = 0.56

Elliptical

Lensiform

Wider

Parallel-sided

v/(s) [7-9] J.1 = 8.09 (J = 0.46

Elliptical

Lensiform

Wider

Parallel-sided

v/(s) [7-8.5] J.1 =7.86 (J = 0.36

Oblong-elliptical

Lensiform

Wider

Parallel-sided

(v)/s [0-5] J.1 = 2.5 (J = 1.75

Oblong-elliptical

Rhombic

Narrower

Parallel-sided, slightly constricted near central nodule

Elliptical

Rhombicllensiform

± Same

Convex, clearly constricted near central nodule

(v)/s [0.5-4] J.1 = 2.01 (J = 0.61

Oblong-elliptical

Concave-rhombic

Narrower I ± same

Parallel-sided, slightly constricted near central nodule

(v)/s [1-3.5] J.1 =2.00 (J = 0.90

Elliptical

Concave-rhombic

Wider

Parallel-sided, slightly constricted near central nodule

v [9-10] J.1 =9.75 (J = 0.38

Elliptical

Concave-rhombic

Wider

Parallel-sided, slightly constricted near central nodule

f/(s) [9-10] J.1 =9.65 (J = 0.33

Oblong-elliptical

Rhombic/(lensiform)

Narrower I ± same

Parallel-sided

J.1 =0.00 (J = 0.00

Hooked [1.5-2]

f[lO]

Oblong-elliptical

Rhombic/(lensiform)

J.1 = 10.00 (J = 0.00

[0.795-0.831 ] J.1 = 0.8135, (J = 0.011

Wider

J.1 = 1.86 (J = 0.23

Parallel-sided, clearly constricted near central nodule

Hooked [2]

vis [6.5]

Elliptical

Concave rhombic

± Same

Parallel-sided, slightly constricted near central nodule

Elliptical

Concave rhombic

Narrower

Parallel-sided

1 Length (pm)

2 Width (pm)

Str./IOpm Shape CRE

41.3-73.3 J.1 = 54.61 (J = 6.62

20.5-31.5 J.1 = 27.47 (J=2.14

6.1-7.3 J.1 =6.54 (J = 0.20

44.3-94.3 J.1 = 54.82 (J = 8.80

26.0-37.8 J.1 = 29.58 (J = 2.35

6.4-7.7 J.1 = 7.21 (J = 0.29

J.1 = 1.95 (J= 0.14

41.6-81.7 J.1 = 53.02 (J = 8.38

27.6-41.1 J.1 = 34.02 (J = 2.82

8.6-9.9 J.1 = 9.29 (J = 0.31

J.1 = 1.97 (J = 0.17

31.4-65.9 J.1 = 43.89 (J = 6.48

19.8-28.9 J.1 = 24.98 (J = 1.88

8.8-10.4 J.1 = 9.66 (J = 0.33

47.1-75.6 J.1 = 57.66 (J = 7.76

26.7-36.7 J.1 = 31.14 (J = 2.75

9.2-9.9 J.1 = 9.59 (J = 0.21

45.7-64.0 J.1 = 56.75 (J = 5.60

27.3-35.6 J.1 = 32.87 (J = 2.74

7.3-8.0 J.1 = 7.59 (J = 0.26

Slightly asymm. [0-1] (f)/s [0-2] J.1 =0.88 (J = 0.35

J.1 = 1.00 (J= 0.76

[0.785-0.814] J.1 = 0.7995, (J = 0.009

21.7-55.5 J.1 = 31.36 (J = 6.11

12.8-21.4 J.1=16.l3 (J = 1.64

10.6-13.4 J.1 = 12.22 (J = 0.68

Hooked [1.5-2]

33.9-52.5 J.1 = 40.14 (J = 5.26

18.0-23.6 J.1 = 19.94 (J= 1.61

9.5-10.7 J.1 = 10.39 (J = 0.35

33.8-53.0 J.1 = 44.40 (J = 5.26

14.8-19.6 J.1 = 18.40 (J = 1.55

10.1-11.6 J.1 = 10.88 (J = 0.51

44.1-82.5 J.1 = 57.70 (J=8.16

26.0-34.7 J.1 = 30.18 (J = 2.17

9.1-9.8 J.1 = 9.39 (J = 0.14

44.8-82.7 J.1 = 58.10 (J = 10.83

25.5-34.9 J.1 = 29.12 (J = 2.51

8.8-10.5 J.1 = 9.89 (J = 0.39

1

51.3

20.0

10.1

I

43.0

16.1

n

4

IV 0\

0

~ ~

~

0

:
98

2

3

4

5

6

7

8

9

10

11

A

B

66

44

71

17

8

100

9

8

91

21

Rounded [0-0.5] J.1 =0.01 (J = 0.05

Hooked [1.5-2]

Hooked [1-2]

Hooked [1-2] J.1 = 1.93 (J = 0.23

Hooked [2] J.1 =2.00 (J = 0.00

J.1 = 1.98 (J = 0.10

Rounded [0-0.5] J.1 =0.06 (J = 0.17

Hooked [1-2] J.1 = 1.69 (J = 0.46

Rounded [0]

[0.787-0.837] J.1 = 0.8114, (J = 0.011 [0.766-0.817] J.1 = 0.7842, (J = 0.010 [0.778-0.806] J.1 = 0.7882. (J = 0.006 [0.790-0.836] J.1 = 0.8050, (J = 0.009 [0.811-0.848 J.1 = 0.8308, (J = 0.011

[0.783-0.844] J.1 = 0.8083, (J = 0.012 [0.786-0.805] J.1 = 0.7961, (J = 0.006 [0.786-0.804] J.1 = 0.7946, (J = 0.006 [0.819-0.861 ] J.1 = 0.8386, (J = 0.009

[0.793]

12.3

Rounded [0]

vis [4]

[0.785]

0 0

'"

Morphological Variation in Diploneis smithii and D. fusca

58

14

...

13

~

12

111

s:I

11

'C

10

Gl

--... ...'" s:l

o

9

III

8

rt.I

:;

(I

.= ... ........

--:...a a

I1.l

.....''""

1.0

o

..

o

o

II

0

D

o

o

7

0.5

o

o

D

110 II

.cl

2

9

3

4

5

6

7

8

9 1011

A

o

0

: ~B ~

: "

0

0

0

0.0

o

1

2

3

~~g

01----r-----.-.---.---¥---=;:----.-,.---.-..------,----,----, 1234567891011 A B

Ilorphotype

4

5

6

7

8

9 10 11 A B

Ilorphotype

60

o

7

lD

B

8

4

000

I'l

10

III

1.5

110 II

Ilorphotype

•

D

.cl

1

(I

I

D

5-l--~~~,-..-----.-~~--,,-.----.-----.--r--r

...'=" ...'= •"

59 2.0

I'l

6

II

••I'l

261

0.87 0.86 0.85 0.84 0.83 0.82 0.81 0.80 0.79 0.78

61

o o

0.77

0.76 0.75 +----.--r---r--,r--.--.---,--,------.---r-----r-,-, 1234567891011 A B

Ilorphotype

Figs. 58--61. Box plots of scored characters against morphotype; boxes enclose 25th to 75th percentiles (50th percentile marked within box); caps indicate 10th and 90th percentiles; all outliers shown beyond caps. Fig. 58: Striation density vs morphotype; Fig. 59: Shape of central raphe endings vs morphotype; Fig. 60: Valve structure (tenths of total as fUsca-structure) vs morphotype; Fig. 61: Rectangularity vs morphotype. tudinal canals about the same width as central nodule. Raphe-sternum furrow slightly convex, clearly constricted close to the central nodule. Central external raphe endings slightly asymmetrical, but not hooked (Fig. 44).

Morphotype 7 (Figs. 21-24,45; 100 valves measured): Small- to medium-sized finely-striate, oblong-elliptical diatoms. "vacillans" -structure occupying about onefifth of the valve surface distal to the longitudinal canals (Fig. 45). Longitudinal canals and raphe system forming a narrowly concave-rhombic shape. Longitudinal canals narrower than or about the same width as central nodule. Raphe-sternum furrow parallel-sided, slightly constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Fig. 45).

Morphotype 8 (Figs. 25-27, 46; 9 valves measured): Small- to medium-sized finely-striate, elliptical diatoms. "vacillans" -structure occupying about one-fifth of the valve surface distal to the longitudinal canals (Fig. 46). Longitudinal canals and raphe system forming a narrowly concave-rhombic shape. Longitudinal canals wider than central nodule. Raphe-sternum furrow parallel-sided, slightly constricted close to the central nodule. Central external raphe endings round, bilaterally symmetrical (Fig. 46). Morphotype 9 (Figs. 28-30, 47; 8 valves measured): Small- to medium-sized finely-striate, elliptical diatoms. "vacillans" -structure occupying virtually the whole valve surface distal to the longitudinal canals (Fig. 47). Longitudinal canals and raphe system forming a narrowly concave-rhombic shape. Longitudinal canals

262

s. J. M. DROOP

wider than central nodule. Raphe-sternum furrow parallel-sided, slightly constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Fig. 47). Morphotype 10 (Figs. 33-35,48,50-53,55; 91 valves measured): Medium- to large-sized rather coarselystriate, oblong diatoms. ''jusca''-structure occupying virtually the whole of the valve surface distal to the longitudinal canals (Fig. 48). Longitudinal canals and raphe system usually forming a narrowly rhombic shape. Longitudinal canals narrower than (rarely about the same width as) central nodule. Raphe-sternum furrow parallel-sided, not noticeably constricted close to the central nodule. Central external raphe endings round, bilaterally symmetrical (Figs. 48, 50-53, 55). Morphotype 11 (Figs. 36-38,49,56; 21 valves measured): Medium- to large-sized rather coarsely-striate, oblongelliptical diatoms. ''jusca''-structure occupying the whole of the valve surface distal to the longitudinal canals (Fig. 49). Longitudinal canals and raphe system forming a narrowly rhombic (rarely lensiform) shape. Longitudinal canals wider than central nodule. Raphesternum furrow parallel-sided, clearly constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical (Figs. 49, 56). Unmatched diatom A (Fig. 31; 1 valve measured): Medium-sized finely-striate, elliptical diatom. "vacillans"-structure occupying about two-thirds of the valve surface distal to the longitudinal canals. Longitudinal canals and raphe system forming a narrowly concaverhombic shape. Longitudinal canals about the same width as the central nodule. Raphe-sternum furrow parallel-sided, slightly constricted close to the central nodule. Central external raphe endings hooked, bilaterally asymmetrical. Unmatched diatom B (Fig. 32; 1 valve measured): Small finely-striate, elliptical diatom. "vacillans"-structure occupying about four-tenths of the valve surface distal to the longitudinal canals. Longitudinal canals and raphe system forming a linear and more or less parallelsided shape, slightly expanded close to central nodule. Longitudinal canals narrower than the central nodule. Raphe-sternum furrow parallel-sided, slightly constricted close to the central nodule. Central external raphe endings round, bilaterally symmetrical. In summary, a qualitative analysis of the data suggests that all 11 morphotypes can be clearly distinguished from each of the others using a combination of morphometric and qualitative characters. Even pairs of morphotypes that are superficially rather similar can be separated with confidence:

Morphotypes 1 and 2 (Figs. 2-4, 5-7) are clearly separated by a combination of shape of central raphe endings (Fig. 59), microstructure of valve surface (Fig. 60), shape of valve outline (Fig. 61), shape of longitudinal canals (Table 2), and to a lesser extent striation density (Fig. 58). Morphotypes 1 and 6 (Figs. 2-4, 18-20) are well separated by shape of central raphe endings (Fig. 59), microstructure of valve surface (Fig. 60), and shape of longitudinal canals (Table 2). Morphotypes 3 and 4 (Figs. 8-10, 11-14), which are very similar in many respects, are separated by a combination of length and width (Fig. 57), and by shape of valve outline (Fig. 61). Morphotypes 7-9 (Figs. 21-24, 25-27, 28-30) are easily separated by a combination of shape of central raphe endings (Fig. 59), microstructure of valve surface (Fig. 60) and shape of valve outline (Fig. 61). Morphotypes 10 and 11 (Figs. 33-35, 36-38) are separated by shape of central raphe endings (Fig. 59), shape of valve outline (Fig. 61), and shape of longitudinal canals (Table 2). Morphotypes 5 and 10, which share a similar outline and shape of longitudinal canals, are distinguished by their different shape of central raphe endings (Fig. 59) and different stria structures (Fig. 60). Valve A (Fig. 31) is very similar to Morphotype 9 (Figs. 28-30), except for a markedly different valve surface microstructure (Fig. 60). It is likely that Valve A represents simply an aberrant individual of Morphotype 9. Valve B (Fig. 32) is similar to Morphotypes 7-9 (Figs. 21-30) with respect to its size and fineness of structure, but differs in its narrow valve and in the narrowness of its longitudinal canals. Its central raphe endings are straight. The narrowness of its longitudinal canals recalls D. littoralis (and the shape of the valve, D. littoralis var. clathrata (OSTRUP) CLEVE), but in D. littoralis the longitudinal canals are not expanded around the central nodule; nor are the central raphe endings straight.

Principal components analysis The following characters were used in the PCA: Length, width, striation density, shape of central raphe endings, ''jusca/vacillans: smithii" ratio on valve surface, and rectangularity. The results are shown in Figs. 62-64 and Table 3. Envelopes linking the outliers of each morphotype have been superimposed to show the extent to which the PCA agrees with the groupings already defined above. Table 3 shows that the first three principal components account for 89 % of the total variation, the fourth 7.3 %, and PCs 5 and 6 2.2% and 1.4% respectively. On this basis, plots were prepared showing each of the first four

Morphological Variation in Diploneis smithii and D. fusca

62

3

N

U

P.

2

0

o

-1

-1

-2

-2

-3

-5

-4

-3

-2

a

-1

234

63

3

2

- 3 '-------'-_--'-_-'------l_--'-_--'-_-'------l_--' -5 -4 -3 -2 -1 0 234

PC 1

PC 1

64

3

• ... • ... • o

Morphotype Morphotype Morphotype Morphotype Morphotype Morphotype 'V Morphotype o Morphotype 6 Morphotype o Morphotype o Morphotype

2

~

u

p.,

a -1

1 2 3 4 5 6 7 8 9 10 11

• Valve A T Valve 8

-2 -3 -3

-2

-1

0

2

3

PC 2

Figs. 62-64. Principal Components Analysis plots. Envelopes enclose outliers of each morphotype. Fig. 62: PC 2 vs PC 1; Fig. 63: PC 3 vs PC 1; Fig. 64: PC 3 vs PC 2. Table 3. Summary of the Principal Components Analysis: Eigenanalysis of the correlation matrix. Eigenvalue Proportion Cumulative

2.9647 0.494 0.494

1.3033 0.217 0.711

1.0787 0.180 0.891

0.4361 0.073 0.964

0.1343 0.022 0.986

0.0829 0.014 1.000

Character

PC 1

PC2

PC3

PC4

PCS

PC6

1 2 3 4 5 6

0.538 0.518 -0.430 -0.357 0.287 0.220

0.097 0.254 -0.406 0.387 -0.184 -0.760

-0.059 -0.221 -0.386 -0.506 -0.735 0.046

-0.437 -0.177 -0.176 -0.540 0.525 -0.423

-0.196 -0.360 -0.683 0.416 0.220 0.379

0.685 -0.677 0.054 -0.043 0.136 -0.223

Length Width Striae / 10 flm ShapeCRE Valve structure Rectangularity

263

264

S. J. M. DROOP

PCs against each other; those involving PC 4 added nothing new to the other three, and so are not illustrated. It is clear from Table 3 that PC 1 is very heavily weighted in favour of length and width. Since these characters usually vary as part of the life-cycle anyway (see Discussion), this result was to be expected. To offset the bias caused by the inclusion of length and width, whose variation tends to have only special, restricted taxonomic significance, the same analysis was performed on the four non-dimensional characters to try to obtain better separation of the morphotypes. The results of this analysis are not shown, since the improvement was minimal, and because an analysis involving six characters seems intuitively more sound than one involving only four. The best plots for separating the morphotypes are PC 3 against PC 1 (Fig. 63) and PC 3 against PC 2 (Fig. 64). The plot of PC 2 against PC 1 (Fig. 62), although showing an undifferentiated central area with many envelopes, does show good separation of Morphotypes 2, 7 and 10 from most of the others. In summary, the following envelopes can be completely separated using a combination of Figs. 62-64: 2, 5, 8, 10 and 11. The following pairs of morphotypes cannot reliably be separated on the basis of the PCA (although they can all be distinguished by individual characters (Figs. 57-61) and by eye): 1 and 6,3 and 4, and 4 and 9. Thus, out of a total of 55 possible pairwise comparisons, only three fail to give complete separation. However,

even in these three the overlap is minimal: in each case only one valve is within the envelope of the other morphotype. Fig. 62 shows one valve of Morphotype 3 within the envelope of Morphotype 4; Fig. 64 shows one valve of Morphotype 6 within the envelope of Morphotype 1, and one valve of Morphotype 9 within the envelope of Morphotype 4. The single unmatched valve A (Fig. 31) is within or very close to Morphotype 4 in Figs. 62-64, but it is unlikely that the relationships of Valve A are in that direction (compare Fig. 31 with Figs. 11-14). It has been suggested, above, that its relationships are with Morphotype 9. Valve B (Fig. 32) is within the envelopes of Morphotypes 7 and 9 (Fig. 62), or very close to Morphotype 7 (Figs. 63, 64) or very close to Morphotype 5 (Fig. 64). Qualitative analysis suggests that its true relationships may be further afield.

Hierarchical Cluster Analysis The same data as were used in the PCA (i.e. characters 1-6 in Table 2) were subjected to Hierarchical Clustering Analysis and the resulting dendrogram is shown in Fig. 65. Table 4 shows how the clusters formed at a particular level of dissimilarity (line X in Fig. 65) agree with the morphotypes delimited empirically. The correlation coefficient between the dissimilarity matrix and the

Table 4. Agreement between groups defined by Hierarchical Cluster Analysis and those defined empirically. Numbers in table indicate how many valves of each morphotype occur in each HCA cluster (as defined by line X in Fig. 65). Columns are HCA clusters; rows are morphotypes. Col. n is number of valves in each morphotype; col. Mis morphotype number.

M

n 98

1

66

2

44

3

71

4

17

5

8

6

100

7

9

8

8

9

91

10

21

11

1

A

1

B

a

b

c

d

f

e

g

i

h

j

k

I

m

n

0

p

98 63 39

4 70

1

15 1

2

1

2

7

96

4

9 7

1

91 11

10

1 1

Morphological Variation in Diploneis smithii and D. fusca

2. 0

Dissimilarity

1.5

1.0

0.5

265

0.0

Fig. 65. Hierarchical Clustering Analysis dendrogram. Clustering method: UPGMA, from the dissimilarity matrix. All 535 valves are included, but not labelled for the sake of clarity. Line X defines Clusters a-po See Table 4 for a comparison with Morphotypes 1-11.

266

S. J. M. DROOP

cophenetic value matrix (calculated in reverse from the dendrogram) is 0.83, implying a high degree of reliability of the dendrogram. Fig. 65 shows the whole dendrogram, but the OTUs are not labelled individually for the sake of simplicity. The dendrogram suggests that Morphotypes 1 and 6 (more or less represented by Clusters a and b in Fig. 65) are rather closely related to each other, as are Morphotypes 2, 3 and 4 (see Clusters c-e), although these include some valves of Morphotypes 9 and 11; of the other clusters in the central part of Fig. 65, only Morphotype lOis separated wholly and without any other valves (Cluster j). At the bottom of Fig. 65 is a large cluster containing most of Morphotypes 7 and 8, along with two other valves (see Clusters n-p); these are separated at quite a high level from the rest of the dendrogram, and a long way from Morphotype 9, which they most closely resemble qualitatively (see part of Cluster e). The position of line X in Fig. 65 was chosen arbitrarily: it represents the position where it is easiest to test the agreement between the statistical and qualitative analyses. It defines more clusters than there are morphotypes, but to have moved the line slightly to the left (so as to reduce the number of clusters) would have meant combining the bulk of Morphotypes 2 and 3, and, a bit further, Morphotypes 1 and 6, as well as parts of 4, 5, 7 and 11. The high level of agreement between the clusters formed by the HCA and the morphotypes already delimited can be seen in Table 4: all 98 valves of Morphotype 1 are included in Cluster a (as well as only one other valve); 63 out of 66 valves of Morphotype 2 in Cluster c, and so on. The worst agreement can be seen for Morphotype 11, which is split almost equally between Clusters e and h; Cluster e includes most of Morphotypes 4 and 9, half of Morphotype 11, and Valve B.

Discussion Comparison between statistical and qualitative analyses The qualitative analysis identified 11 clear-cut morphotypes, and the statistical analyses confirmed those groupings with a few exceptions: Morphotypes 1 and 6; 3 and 4; and 4 and 9 were not completely separated by PCA; and HCA showed small overlaps between several pairs of morphotypes, most notably between Morphotypes 4, 9 and 11. How are these inconsistencies to be viewed, since, qualitatively, the morphotypes appear quite different (see Table 2 and Figs. 2-38)? Incomplete separation of some morphotypes probably occurred because these morphotypes are so similar in most of the characters included in the study that the small number of

differences are averaged out; the many other differences by which they can be recognized by eye were not included, since they are so difficult to measure. It has been shown beyond reasonable doubt by both qualitative and statistical analyses that the null hypothesis (that the observed variation is more or less continuous and has no taxonomic significance) does not stand up to inspection: 11 morphotypes, each represented by a population showing a clear size-reduction sequence, and 2 individual valves that fit none of the morphotypes, have been identified from the representatives of D. smithii sensu lato in a single sympatric sample. But what significance should be placed on such discontinuities and what conclusions can be drawn, given that this is only one sample from one locality?

Variation within morphotypes In order to understand the variation between morphotypes of D. smithii sensu lato, it is necessary first to interpret the variation within the morphotypes. Diatoms reproduce vegetatively through mitotic division, so that each cell can potentially produce a clone of many genetically identical individuals. These individuals are very similar morphologically, except that succeeding generations are usually fractionally smaller (the MACDoNALD-PFITZER rule: MACDONALD 1869, PFITZER 1869; see ROUND et al. 1990). It is conceivable, therefore, that each morphotype present in the E 1014 sample is nothing more than a clone, and that any variation within it is small enough to· be accounted for by phenotypic plasticity or somatic mutation. However, the genetic structure of each morphotype is unlikely to be so simple. All the morphotypes exhibit the kind of variation in length, width and aspect ratio that is usually associated with a sexual life-cycle. The typical diatom life-cycle (GEITLER 1932, 1973; DREBEs 1977; ROUND et al. 1990; MANN 1993) involves long periods of mitotic cell division, when mean cell size decreases, alternating with a much shorter sexual phase, during which the old, small cell walls are discarded and special zygotic cells (auxospores) are produced that swell and so restore cell size to a maximum. It is very rare for size restitution via auxospores to be decoupled from allogamous sexual reproduction. When it is, there may still be the possibility of meiotic recombination, as in automictic species or, exceptionally, all trace of sexual reproduction may be lost. But such cases are very uncommon and generally involve particular races or varieties of otherwise normal sexual species (GEITLER 1973, 1982; MANN 1993). It is also possible for diatoms to avoid size reduction, so that they too are asexual (see WIEDLING 1948, ROUND et al. 1990, MANN 1993 for ex-

Morphological Variation in Diploneis smithii and D. fusca

amples), but this clearly does not apply to the Diploneis morphotypes at Oban. Hence, based on the fact that most diatoms exhibiting a size reduction-restitution cycle are sexual, it is most parsimonious to assume that Diploneis smithii and its allies are also sexual, and that periods of vegetative cell division and size reduction alternate with episodes of allogamous sexual reproduction. No direct evidence of sexual reproduction is available for D. smithii and related forms, but there are data for other Diploneis species (GEITLER 1932). KARSTEN (1899) found zygotes and auxospores in D. didyma (EHRENB.) CLEVE (as Navicula didyma EHRENB.), while PFITZER (1871) observed pairing and auxosporulation in the freshwater species D. elliptica (KUTZ.) CLEVE (as Navicula elliptica KUTZ.); here only one auxospore was found per pair of gametangia. Gamete formation and allogamous reproduction, with the formation of one auxospore, has also been recorded in a freshwater species with elliptical valves (D. G. MANN, unpublished observations), while allogamous reproduction producing two auxospores per pair of gametangia has been found in a marine species similar to D. chersonensis (GRUN.) CLEVE (D. G. MANN & S. J. M. DROOP, unpublished observations). IDEl (pers. comm.) has documented allogamous sexual reproduction in a marine species of Diploneis. Finally, MIQUEL (1893-96) recorded single auxospores being produced, apparently through a sexual process, in a marine Diploneis he called Navicula (= Diploneis) elliptica (D. elliptica is probably strictly freshwater), though automixis cannot be ruled out (GEITLER 1932). Both D. didyma and D. elliptica exist in a number of identifiable morphotypes (DRoop, in prep.), so there is nothing unusual in the pattern of variation shown by D. smithii and its allies to suggest that their reproductive strategy should be anything other than allogamous. If, as seems likely, the Oban Diploneis are sexual forms, then each is probably heterogeneous genetically. For each to be a homogeneous clone, it would have to have arisen very recently, with no opportunity for mutation and recombination to introduce variation. Recent origin is unlikely, however, because of the occurrence of these morphotypes elsewhere besides Oban (see below).

Variation between morphotypes In spite of the evidence presented above, it could still be argued that the 11 morphotypes of D. smithii and its allies are merely different phenotypic expressions of the same genotype. Cox (1993) has summarized the available literature, but it is worth mentioning here the work of SYVERTSEN (1979) who showed that a single clone of Thalassiosira CLEVE could be identified as

267

T. gravida CLEVE or T. rotula MEUNIER depending on the culturing temperature. A similar phenomenon has been noted in wild populations of Stephanodiscus hantzschii GRUN. in Loch Leven (BAILEY-WATTS 1988), where a temporal succession of forms of the species followed changes in nutrient-loading of the loch. That these were phenotypic rather than genotypic variants was shown by the presence of heterovalvar cells. Several examples of morphological polymorphism have been shown in other algae as well (TRAINOR et al. 1971), so the phenomenon cannot be regarded as rare or insignificant. However, for this argument to be applied to the Oban morphotypes of D. smithii sensu lato, it would have to involve both the occurrence of 11 different ecological niches in the unstable environment of the sandy shore where E 1014 comes from, and simultaneous phenotypic adjustment by all the morphotypes to each niche. This seems highly unlikely. In addition, the occurrence of polymorphism from a single genotype would necessitate the production of Janus (heterovalvar) cells - this is simply not possible for these morphotypes, because of differences in shape and aspect ratio. Another possible interpretation is that the differences between the morphotypes could be accounted for by particulate variation, in the same way that MENDEL'S peas were either sugary or starchy, smooth or wrinkled; there were no intermediates because of the biochemical pathways involved. It could be argued, therefore, that the ontogeny of these diatoms only allows the expression of some character states. If this were the case, the whole range of variation encountered within D. smithii sensu lata could be encompassed by one variable sexual species. However, this is unlikely, for the following reasons: firstly, many of the correlated characters that distinguish the morphotypes involve quite different parts of the valve, and it is very hard to imagine that they could be under the same control; secondly, there are valves that do not fit all the character states for a particular morphotype (if the character complexes were under strict unitary control, such mistakes should not occur); thirdly, the same character states occur in different combinations in other diatom species. There is now substantial evidence that several diatom species exist in a number of similar but morphologically distinguishable races that do not interbreed. GEITLER (for example 1968, 1982) demonstrated reproductive isolation between "Sippen" of several common freshwater epiphytic species. Although two races of Cocconeis placentula EHRENB. have dispensed with sex altogether and turned to parthenogenesis (vars. klinoraphis GEITLER and lineata (EHRENB.) VAN HEURCK), all the other races investigated by GEITLER (1982) exhibit normal sexual pairing and auxospore formation. The races can be sympatric and yet are subtly different morphologically, implying reproductive isolation or intense disruptive selection. Only one case of hybridization was docu-

268

s. J. M. DROOP

mented (GEITLER 1958a), however, involving vars. pseudolineata GEITLER and euglyptoides GEITLER. Auxospores were observed in six separate pairings, either between typical pseudolineata and euglyptoides or between typical pseudolineata and a supposed hybrid, while intermediates between these varieties were observed in the population, implying successful hybridization and backcrossing. Similarly, MANN (1984, 1988, 1989) showed the same sort of isolation between morphotypes of some common freshwater epipelic species. Six morphotypes of Sellaphora pupula (KUTZ.) MERESCHK. were identified in one pond, five of which were shown to exhibit normal sexual reproduction, but with pairing only between individuals of a particular morphotype. Sexual reproduction was not witnessed at all in the sixth morphotype and the valves were all very similar in size, so it is tempting to speculate (MANN 1989) that this morphotype avoids size reduction and hence also sex, just as seems to happen also in Caloneis amphisbaena (BORY) CLEVE and Craticula ambigua (EHRENB.) D. G. MANN (MANN 1993). GALLAGHER (1980, 1982) has presented evidence that winter and summer blooms of Skeletonema costatum (GREV.) CLEVE, although apparently identical morphologically, show strikingly different genotype frequencies. This clearly implies barriers to gene flow between the two populations or intense selection. By analogy with the above, the eleven sympatric races identified in D. smithii and its allies probably do not interbreed, and so it is likely that they function as normal allogamous species. In effect, then, one would be applying a biological species concept, whose main criterion would be the ability to interbreed. Such a concept is already well-developed for animals (e.g. MAYR & ASHLOCK 1991) and has been argued for higher plants (MAYR 1992). The biological species concept has its problems, though, in dealing with "deviant" reproductive strategies like automixis and apomixis, or with allochronic and allopatric "species", where the potential for interbreeding cannot be tested except artificially in a laboratory. In addition there are the complications of a taxonomic strategy that does not include morphological dissimilarity as a criterion, since there is no a priori reason why individuals that accept or reject each other on the basis of physiological or chemical recognition systems should also be morphologically dissimilar. There is one aspect of phenotypic plasticity that suggests that the arguments for a biological species concept must be viewed with caution: if the expression of genes for morphology can be altered by environmental effects, why not also the expression of genes for physiology and behaviour, such as the recognition of "self' in potential mating partners? However, I am not aware of any instances where environmental control of recognition systems has been shown to occur in microalgae.

Implications for diatom systematics The evidence presented here for the recognition of 11 morphotypes of D. smithiiljusca is not enough on which to base the description of new species: it represents only a single point in time and space. My findings are not backed up by breeding studies, although there is a striking similarity between the levels of variation found in these species with those seen in the freshwater species studied by MANN and GEITLER. This suggests at least the possibility that a similar phenomenon is at work here, and future observations may confirm the reproductive barriers implied by the morphological evidence. What the evidence does show quite clearly, though, is that variation within this group of diatoms is not continous within sympatric populations, and that patterns of variation in several characters are correlated, so that discontinuities can be observed and documented. The following additional evidence is relevant: 1) Another sample, EI016, taken from the same beach at the same time as E 10 14 has most of the same morphotypes, but in different proportions; Morphotype 6 (very rare in E 1014) is much more common in E 1016, even though it was collected only 50 m or so away at the same level on the beach, but from sand that had a lower content of shell fragments. 2) Each of the morphotypes has been found in other sites around the British coast (where there are also additional distinct morphotypes not encountered in E 1014 and so not described here). The other populations of each morphotype are not always identical with those in E 1014 in every respect, especially in continuously variable characters, but most of the characters by which the morphotypes are recognized are constant. 3) Some of the morphotypes can be recognized in published illustrations (Table 5). Few specific localities are given (the most distant is Villefranche, in southern France), but it is unlikely that any of the diatoms illustrated were from Oban, where E 1014 was collected, since Oban is not a classical collecting site for diatomists. Other illustrations could have been included in Table 5, but since many morphotypes can only be distinguished on the basis of microstructure, illustrations are listed only where there was virtually no doubt as to their identity. It is not the purpose of this paper to attempt to name the morphotypes or to resolve infraspecific synonymy, but it is likely that some morphotypes correspond to taxa that have been named and described before, as varieties or forms. There is a strong argument for recognizing the morphotypes taxonomically. They probably represent normally-functioning sexual species that show slightly different ecological preferences, resulting in their different frequencies at different sites. Even if they are merely different ecotypes of a single species, the formal recognition of the morphological variation,

Morphological Variation in Diploneis smithii and D. fusca

269

Table 5. Occurrence of identifiable morphotypes in published illustrations. M'type

Author, date

Page / Fig., etc.

Species

Origin

3

PERAGALLO&PERAGALLO (1897-1908)

Plate 20, Fig. 9

N. fusca var. delicata CLEVE

Villefranche, France

4

SCHMIDT (1874-1959)

Plate 7, Fig. II

Navicula?

Cresswell, UK

5

HUSTEDT (1927-1966)

Fig. 1059, (b)

D. fusca var. aestiva with "smithii" structure

Europe

9

HUSTEDT (1927-1966)

Fig. 1060, (c)

D. vacillans

Europe

HENDEY (1964)

Plate 32, Fig. 4

D. fusca (GREG.) CLEVE

UK

10

coupled with an analysis of precise ecological preferences, would give badly-needed fine tuning in archaeological and palaeoecological studies, where past conditions are reconstructed on the basis of knowledge of the conditions favoured by extant diatoms. The occurrence of identifiable morphotypes within diatom species is much more common than was previously thought. Of the British species of Diploneis, for example, most exist as two or more recognizable and constant morphotypes (DROOP, in prep.). The D. smithii sensu lato complex probably includes about 30, including those described above; the following are some other examples: D. nitescens (GREG.) CLEVE (2), D. suborbicularis (GREG.) CLEVE (2), D. coffaeiformis (A. S.) CLEVE (3), D. notabilis (GREY.) CLEVE (3), D. papula (A. S.) CLEVE (ca. 6), D. littoralis (2), D. didyma (3), D. bombus (EHRENB.) EHRENB. ex CLEVE (3), D. interrupta (KUTZ.) CLEVE (2), D. incurvata (GREG.) CLEVE (3), D. stroemii HUSTEDT (2), and D. crabro (EHRENB.) EHRENB. ex CLEVE, (ca. 5). Some have been described previously, but of these, most are now regarded as varieties or reduced to synonymy (HARTLEY 1986, HUSTEDT 1927-1966). Distinct sympatric morphotypes have also been observed in Lyrella KARAYEVA and Hantzschia GRUN. (MANN, in preparation) and Petroneis A. J. STICKLE & D. G. MANN (JONES, in prep.). Published occurrences of this phenomenon involve Sellaphora pupula and Cocconeis placentula already mentioned; Caloneis silicula (EHRENB.) CLEVE, Neidium amplicatum (EHRENB.) KRAMMER and Cymatopleura solea (BREB. & GODEY) W. SMITH (MANN 1989); Navicula cryptocephala KUTZ. (GEITLER 1958b); several species of Gomphonema EHRENB. (GEITLER 1958b, 1963, 1968, 1969, 1970, 1972); Surirella ovata KUTZ., Eunotiaflexuosa (BREB. in KUTZ.) KUTZ., several species of Cymbella AGARDH, Nitzschia dissipata (KUTZ.) GRUN. and Amphora ovalis (KUTZ.) KUTZ. (GEITLER 1968). There is a distinct possibility, therefore, that this phenomenon is widespread, if not universal, among diatoms.

Acknowledgements: Part of this work was done with the help of a NERC grant (GR317923) and I am grateful for their financial support. My thanks, too, to HELEN JONES, FRANK ROUND and EILEEN Cox for many formative discussions. Special thanks are due to MARTIN PULLAN for help with the computing aspects of image-analysis, and to TONY HUNTER of the Scottish Agricultural Statistics Service for his invaluable advice on the statistical analyses. My special thanks to DAVID MANN for his support and encouragement, and for his rigorous review of the manuscript at several stages.

References BAILEYWATTS, A. E. (1988): Studies on the control of the early spring diatom maximum in Loch Leven 1981. In: F. E. ROUND (ed.), Algae and the aquatic environmentcontributions in honour of J. W. G. Lund, C. B. E., F. R.S., pp. 53-87. Bristol. CLEVE, P. T. (1894): Synopsis of the Naviculoid diatoms. Kongl. Svensk. Vetenskaps-Akad. Handl., Part I, Bd. 26: 1-194. CLEVE-EULER, A. (1953): Die Diatomeen von Schweden und Finnland. Kungl. Svensk. Vetenskapsakad. Handl., Part III, Bd. 4 (5): 1-255. Cox, E. 1. (1993): Diatom systematics - a review of past and present practice and a personal vision for future development. Nova Hedwigia, Beih. 106: 1-20. DONKIN, A. S. (1858): On the marine Diatomaceae of Northumberland, with a description of eighteen new species. Trans. Micr. Soc. Lond., n. s. 6: 12-34. DREBES, G. (1977): Sexuality. In: D. WERNER (ed.), The biology of diatoms (Bot. Monogr. 13); pp. 250-283. Oxford. DROOP, S. J. M. (1993): The "Striatometer" - a new device for measuring striation densities: research note. Diatom Research 8 (1): 195-198. EATON, J. W. & Moss, B. (1966): The estimation of numbers and pigment content in epipelic algal populations. Limnol. & Oceanogr. 11 (4): 584-595. GALLAGHER, J. (1980): Population genetics of Skeletonema costatum (Bacillariophyceae) in Narragansett Bay. 1. Phycol. 16: 464-474.

270

S. J. M. DROOP

- (1982): Physiological vanatlOn and electrophoretic banding patterns of genetically different seasonal populations of Skeletonema costatum (Bacillariophyceae). J. PhycoI. 18: 148-162. GEITLER, L. (1932): Der Formwechsel der pennaten Diatomeen (Kieselalgen). Arch. Protistenkd. 78: 1-226. - (1958a): Fortpflanzungsbiologische Eigenttimlichkeiten von Cocconeis und Vorarbeiten zu einer systematischen Gliederung von Cocconeis placentula nebst Beobachtungen an Bastarden. Osterr. Bot. Zeitschr. 105: 350-379. - (1958 b): Notizen tiber Rassenbildung, Fortpflanzung, Formwechsel und morphologische Eigenttimlichkeiten bei pennaten Diatomeen. sterr. Bot. Zeitschr. 105: 408-442. - (1963): Kurze Mitteilung: Rassenbildung bei Gomphonema angustatum. Osterr. Bot. Zeitschr. 110: 481-483. - (1968): Kleinsippen bei Diatomeen. Osterr. Bot. Zeitschr. 115: 354-362. - (1969): Notizen tiber die Auxosporenbildung einiger pennater Diatomeen. Osterr. Bot. Zeitschr. 117: 265-275. - (1970): Kleinsippen von Gomphonema angustatum. Osterr. Bot. Zeitschr. 118: 197-200. - (1972): Sippen von Gomphonema parvulum. Paarungsverhalten und Variabilitat pennater Diatomeen. sterr. Bot. Zeitschr. 120: 257-268. - (1982): Die infraspezifischen Sippen von Cocconeis placentula des Lunzer Seebachs. Arch. Hydrobiol., SuppI. 63 (1): 1-11. GILMOUR, J. S. L. & HESLOP-HARRISON, J. (1954): The deme terminology and the units of micro-evolutionary change. Genetica 27: 147-161. GREGORY, W. (1857): On some new forms of marine Diatomaceae, found in the Firth of Clyde and in Loch Fine. Trans. Roy. Soc. Edinb. 21(4): 473-542. HARTLEY, B. (1986): A check-list of the freshwater, brackish and marine diatoms of the British Isles and adjoining coastal waters. J. Marine BioI. Assoc. U. K. 66: 531-610. HENDEY, N. I. (1964): An introductory account of the smaller algae of British coastal waters. Fishery Investigations Series, 4: Part 5: Bacillariophyceae (Diatoms). London. HUSTEDT, F. (1927-66): Die Kieselalgen Deutschlands, Osterreichs und der Schweiz unter Beriicksichtigung der tibrigen Lander Europas sowie der angrenzenden Meeresgebiete. In: Dr. Rabenhorst's KryptogamenFlora von Deutschland, Osterreich und der Schweiz 7. Leipzig. KARSTEN, G. (1899): Die Diatomeen der Kieler Bucht. Wiss. Meeresunters., Kie14: 19-205. KOTZING, F. T. (1844): Die Kieselschaligen Bacillarien oder Diatomeen. Nordhausen. MACDONALD, J. D. (1869): On the structure of the diatomaceous frustule and its genetic cycle. Ann. Mag. Nat. Hist. Ser. 4, 3: 1-8. MANN, D. G. (1984): Observations on copulation in Navicula pupula and Amphora ovalis in relation to the nature of diatom species. Ann. Bot. 54: 429-438. - (1988): The nature of diatom species: analysis of sympatric populations. In: F. E. ROUND (ed.), Proceedings of

o

o

the ninth International Diatom Symposium, Bristol, August 24-30, 1986, pp. 317-327. Bristol and Koenigstein. - (1989): The species concept in diatoms: evidence for morphologically distinct, sympatric gamodemes in four epipelic species. PI. Syst. Evol. 164: 215-237. - (1993): Patterns of sexual reproduction in diatoms. In: H. VAN DAM (ed.), Proceedings of the Twelfth International Diatom Symposium, held at Renesse, The Netherlands, 30. August-5. September 1992. Hydrobiologia 269-270 (Developm. in Hydrobiol. 90): 11-20. MAYR, E. (1992): A local flora and the biological species concept. Am. 1. Bot. 79 (2): 222-238. - & ASHLOCK, P. D. (1991): Principals of systematic zoology. 2nd edition. New York. MIQUEL, P. (1893-96): Du n~tablissement de la taille et de la rectification de la forme chez les Diatomees. La Diatomiste 16: 61-69; 17: 88-98. PERAGALLO, H. & PERAGALLO, M. (1897-1908): Diatomees marines de France et des districts maritimes voisins. Grez-sur-Loing. PFITZER, E. (1869): Ober Bau und Zellteilung der Diatomeen. Bot. Zeitung 27: 774-776. - (1871): Untersuchungen tiber Bau und Entwicklung der Bacillariaceen (Diatomaceen). Bonn. ROUND, F. E., CRAWFORD, R. M. & MANN, D. G. (1990): The diatoms: biology & morphology of the genera. Cambridge. SCHMIDT, A. (1874-1959): Atlas der Diatomaceen-Kunde, begriindet von Adolf Schmidt, fortgesetzt von Martin Schmidt, Friedrich Fricke, Heinrich Heiden, Otto Miiller und Friedrich Hustedt. Plates 1-460. Reprinted 1984 by Koeltz, Koenigstein. SMITH, W. (1853): A synopsis of the British Diatomaceae; with remarks on their structure, functions and distribution; and instructions for collecting and preserving specimens. Vol. I. London. - (1856): A synopsis of the British Diatomaceae; with remarks on their structure, functions and distribution; and instructions for collecting and preserving specimens. Vol. II. London. SNEATH, P. H. A. & SOKAL, R. R. (1973): Numerical taxonomy. San Francisco. SYVERTSEN, E. (1979): Thalassiosira gravida and T. rotula: ecology and morphology. Nova Hedwigia, Beih. 54: 99-112. TRAINOR, F. R., ROWLAND, H. L., LYLIS, J. C., WINTER, P. A. & BONANOMI, P. L. (1971): Some examples of polymorphism in algae. Phycologia 10(1): 113-119. VANLANDINGHAM, S. L. (1969): Catalogue of the fossil and recent genera and species of diatoms and their synonyms. Part III. Lehre. WIEDLING, S. (1948): Beitrage zur Kenntnis der vegetativen Vermehrung der Diatomeen. Bot. Notiser 3: 322-354. Accepted: February 18, 1994. Author's address: STEPHEN J. M. DROOP, Royal Botanic Garden Edinburgh, Inverleith Row, Edinburgh EH3 5LR, U. K.