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Nova Hedwigia, Beiheft 142, 027–049 Stuttgart, September 2013

Article

Mallomonas pumilio group (Chrysophyceae/ Stramenopiles) – a revision based on the scale/scalecase morphology and analysis of scale shape Yvonne Němcová*, Jana Kreidlová, Martin Pusztai & Jiří Neustupa Dept. of Botany, Charles University in Prague, Benatska 2, 128 01 Prague, Czech Republic * Corresponding author: [email protected]

With 12 figures and 1 table

Abstract: The species of the Mallomonas pumilio group belong to the smallest taxa of the genus (whole cells reach a length of ca. 12 –14 μm). This study is based on 244 silica scales of taxa sampled world-wide. Detailed scale and scale-case morphology was investigated by transmission and scanning microscopy and scales were sorted into groups representing species and/or varieties. To evaluate the slight differences in scale morphology we used the methods of landmark-based geometric morphometrics (GM). Homologous points (landmarks) were delimited to represent maximally the scale shape within each unit. Multivariate statistical procedures were used to test differences in shape between a priori set units. The new species M. jubata was described and characterized. The varieties Mallomonas pumilio var. silvicola and M. pumilio var. munda were erected to the species level M. silvicola and M. munda, respectively. The nominal variety of M. pumilio was specified and the emended definition provided. Mallomonas directa, until now not distinguished from M. pumilio var. pumilio, was separated on the bases of larger scales, different scale morphology and scale shape. The new varieties M. pumilio var. dispersa and M. solea-ferrea var. irregularis were introduced. However, molecular data are needed to evaluate whether these taxa should be treated as varieties or separate species. So far, none of the members of Mallomonas pumilio group has been sequenced. Key words: Chrysophyceae, Mallomonas directa, M. jubata, M. munda, M. pumilio var. dispersa, M. pumilio var. pumilio, M. silvicola, M. solea-ferrea var. irregularis, new species, scale morphology, geometric morphometrics

Introduction The Mallomonas pumilio group comprises species with small ovoid to ellipsoidal cells from the section Torquatae with a reticulated scale-shield pattern. The meshes of the reticulum are regularly or irregularly arranged and each encloses one to several pores at the bottom. Mallomonas pumilio Harris & Bradley was described by Harris & Bradley (1957; see their Figs 3, 4 – Plate 3) from the Reading district, England. However, Asmund et al. (1982) demonstrated that their photographs of carbon replicas represent two different species distinguishable by the shape of the collar scales. They designated their Fig. 4 as a lectotype of Mallomonas pumilio var. pumilio © 2013 J. Cramer in Gebr. Borntraeger Verlagsbuchhandlung, Stuttgart, Germany

www.borntraeger-cramer.de

1438-9134/2013/0142-0027 $ 5.75

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Fig. 1. A) Mallomonas pumilio var. pumilio published in Harris & Bradley (1960; their Fig. 60). B) M. pumilio var. pumilio in Asmund et al. (1982); described as M. directa in this paper. C) M. pumilio var. pumilio in Asmund et al. (1982); described as M. pumilio var. dispersa in this paper). Reprinted with permission.

Harris & Bradley emend. Asmund, Cronberg & Dürrschmidt, and described their Fig. 3 as a new species Mallomonas alata Asmund, Cronberg & Dürrschmidt. In 1960, two more figures designated as M. pumilio were published by Harris & Bradley (1960) from the same region (Reading district, England). Their Fig. 60 showed a transmission electron microscopy photograph of M. pumilio, whereas their Fig. 63 again showed M. alata f. alata. Asmund et al. (1982) undertook a revision of the M. pumilio group, which resulted in several taxonomic and nomenclatural changes. A new variety, M. pumilio var. munda Asmund, Cronberg & Dürrschmidt, was established. Moreover, they also provided an emended diagnosis of Mallomonas pumilio var. silvicola Harris & Bradley emend. Asmund, Cronberg & Dürrschmidt. In addition to M. alata f. alata, a new forma, M. alata f. hualvensis Asmund, Cronberg & Dürrschmidt, was introduced. Since the 1990s, silica scales which cannot be assigned to any of the known taxa have been reported (Dürrschmidt 1980, Roijackers & Kessels 1981). The description of Mallomonas pumilio var. pumilio provided by Asmund et al. (1982) comprises more than one morphotype (Fig. 1A–C). Moreover, there are neither strains of the Mallomonas pumilio group available in the world’s algal collections, nor corresponding nucleotide sequences in the databases. In landmark-based geometric morphometrics (GM), spatial information is contained in digitalized coordinates of landmarks, discrete loci that are homologous in all objects in the analysis. Shape analysis begins by removing the non-shape information: location, scale and rotation effects (Bookstein 1991). The entire object can then be mapped as a single point into a space

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Fig. 2. Position of landmarks (squares) and semilandmarks (circles).

with as many dimensions as the number of landmarks multiplied by the number of coordinates describing each landmark. The relative position of landmarks among various objects may be used for multivariate analysis of shape. In silica-scaled chrysophyte research, landmark-based geometric morphometric methods have been implemented to investigate temperature and pH related morphological variation of scales in clonal populations (Řezáčová-Škaloudová et al. 2010, Pichrtová & Němcová 2011, Němcová & Pichrtová 2012), to characterize the patterns of scale shape variation in S. echinulata Korshikov in combined gradients of light and temperature (Němcová et al. 2010), to describe scale shape variation in relation to their position within the scale-case (Neustupa et al. 2010), to emphasize the shape differences between scales belonging to the two distinct varieties Mallomonas striata var. striata Asmund and M. striata var. serrata Harris K. & Bradley D.E. (Neustupa & Němcová 2007) and to compare the scale shape of fossil populations (40 million years old) of Mallomonas insignis Penard and the modern ones (Siver et al. 2013). The aim of this study is to provide a revision of the Mallomonas pumilio group based on the scale/scale-case morphology and geometric morphometric data. In this work, we report the results of our investigations, which indicate that, the Mallomonas pumilio group represents several distinctive morphotypes. Two of them are here designated as new species, Mallomonas jubata and M. directa, and an additional two as new varieties M. pumilio var. dispersa and M. solea-ferrea var. irregularis.

Materials and methods This revision is based on cells and scales collected from various localities (comprising mostly the Czech Republic, France – Aquitaine, and South Africa – Western Cape) in 2005 – 2011. Plankton samples (20 μm mesh net) were combined with water squeezed from the submerged vegetation. Drops of the samples were dried onto Formvar-coated transmission electron microscopy (TEM) grids or onto a piece of aluminum foil for scanning electron microscopy (SEM). The TEM grids were examined with a JEOL 1011 transmission electron microscope. Photomicrographs were obtained using a Veleta CCD camera equipped with image analysis software (Olympus Soft Imaging Solution GmbH). For SEM investigation, the Formvar-coated grid (already observed in TEM) or the piece of aluminum foil were mounted onto an SEM stub with double-sided adhesive

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Fig. 3. A) CVA ordination plot shows individual scale scores along canonical axes. B–C) A collar and a body scale of Mallomonas solea-ferrea. D–E) A collar and a body scale of M. solea-ferrea var. irregularis (morphotype A). F–G) A collar and a body scale of M. pumilio var. pumilio (morphotype D). H–I) A collar and a body scale of M. pumilio var. dispersa (morphotype C). J–K) A collar and a body scale of M. jubata. L–M) A collar and a body scale of M. munda. N–O) A collar and a body scale of M. silvicola. P–Q) A collar and a body scale of M. directa (morphotype B).

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Fig. 4. Scale size (length) of investigated taxa.

carbon tape, coated with gold for 5 min (forming a 3 nm layer) with a Bal-Tec SCD 050 sputter coater and observed with a JEOL 6380LV scanning electron microscope. Descriptions of the new species were based on effectively published illustrations, as it was impossible to preserve a specimen showing the features attributed to the taxon (International Code of Botanical Nomenclature, article 37.5). The detailed scale and scale-case morphology was investigated using TEM and SEM and 244 scales were sorted into eight groups. Natural samples containing a single morphotype were used as a reference. Thirty-two landmarks, including twenty-four semilandmarks, were depicted on each scale (Fig. 2). The side of the scale adjacent to the plasma membrane is smooth, while the upper side is structured. Body scales of the Mallomonas pumilio group are rhomboidal; however left anterior flange is considerably shorter and the rim on the corresponding side is longer, enabling the left and right sides of the scale to be distinguished and the landmarks to be placed consistently. A relative warps analysis (RWA) of the shape data, including a shape variables extraction (Bookstein 1991), was conducted on all silica scales (comprising eight groups representing different morphotypes). Scores for the objects on the first 12 principal component (PC) axes (spanning 99.9 % of the total variation) were used for canonical variate analysis (CVA) in PAST, ver. 2.08 b (Hammer et al. 2001). To test for scale shape differences between individual units, the scores on the first twelve relative warps were used for two-group multivariate permutation tests (10 000 permutations) on the Mahalanobis distance between all group pairs. To determine which morphotype should be identified as a type of Mallomonas pumilio var. pumilio, we used one of the scales shown in Figure 1 A (this paper) and published in Harris & Bradley (1960) (their Fig. 60) and we delimited the same set of landmarks (thirty-two landmarks, including twenty-four semilandmarks) on this scale. We chose the one positioned on the left in the microphotograph (marked by an arrow), because the outline was more easily recognizable. The actual lectotype, represented by a carbon replica (Harris & Bradley 1957; their Fig. 4), was unfortunately not suitable for morphometric analysis. A CVA linear classifier (PAST) was applied to assign a landmarked scale representing Mal-

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lomonas pumilio var. pumilio (Fig. 3) to the group that gave a minimal Mahalanobis distance to the group mean. The Mahalanobis distance was calculated from the pooled within-group covariance matrix, giving a linear discriminant classifier. We subsequently added the scale to each of the eight groups (representing eight different morphotypes). The given and estimated assignments to all eight groups were listed for the scale. In addition, the unit assignment was cross-validated by a leave-one-out cross-validation procedure. The length of the scales was measured in the tpsDig program (Rohlf 2006). A non-parametric Kruskal-Wallis test was undertaken in PAST to identify significant pairwise differences in length among individual units. Results were considered significant if p  0.05.

Results Detailed scale and scale-case morphology of taxa from the Mallomonas pumilio group was investigated by means of TEM and SEM. A total of 244 body scales were photographed and sorted into eight a priori defined groups corresponding to different morphotypes (see Table 1; Fig. 3), three of which belonged to well defined, already described species (Mallomonas pumilio var. munda, M. pumilio var. silvicola and M. solea-ferrea). One morphotype represented a new species with unique morphological characters (e.g. crest-like extension of the dome) and was described as Mallomonas jubata; see the taxonomical part. The other four morphotypes were designated by the letters A, B, C and D.

Scale size Based on scale size, the investigated groups were divided into two groups (Fig. 4). The first group, of larger scales, comprised Mallomonas jubata, M. pumilio var. munda, M. pumilio var. silvicola and morphotype B (M. directa). None of the groups was significantly different in scalesize from the other members of this group; however morphotype B (M. directa) varied considerably in size. The second group, of smaller scales, contained Mallomonas solea-ferrea, morphotypes A (M. solea-ferrea var. irregularis), C (M. pumilio var. dispersa) and D (M. pumilio var. pumilio). M. solea-ferrea had the smallest scales and differed significantly from all other groups (p = 0.018 – 6.3E– 5), morphotype A differed in size solely from morphotype C (p = 0.059) and morphotype C from D (p = 0.107).

Geometric morphometrics Only the body scales were used for analysis and the landmarks were delimited to represent the scale shape maximally. Relative warp analysis (= a principal components analysis (PCA) of the covariance matrix of the partial warp scores) revealed the most important trends in the overall shape variation for the entire data set (all morphotypes treated together). Relative warps (RW) 1– 4 accounted for 79.6 % of the total variation in shape (RW1 41.8, RW2 22.0, RW3 10.8 and RW4 5.0). Shape changes of scales associated with extreme positions along the first three RW axes were visualized as deviations from the mean configuration (Fig. 5). The first relative warp described the change from scales possessing wide flanges and a narrow shield with pores/groups of pores to scales with narrow flanges and a wide, extended shield. Shape changes along the second RW were associated with the overall width of the scale. The third RW emphasized the change from a rhomboidal scale shape (with an unequal length of the corresponding flanges) and extended shield to a more regular shape (with an equal length of the corresponding flanges) and reduced shield.

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pronounced

indistinct indistinct

regularly shaped 5 – 6

3 – 5

4 – 5, in central distinct– scales, confluent inflated rows

single pores, indistinct sometimes arranged in rows

irregularly shaped

irregularly shaped

regularly shaped, in central scales forming rows

regularly/ irregularly shaped

regularly shaped 3 – 5

M. silvicola

M. solea-ferrea

(A) M. solea-ferrea var. irregulare

(B) M. directa

(C) M. pumilio var. dispersa

(D) M. pumilio var. pumilio

usually 3 – 4

pronounced

regularly shaped annular groups of 6 pores

M. munda

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distinct– inflated

pronounced

1– 4

regularly/ irregularly shaped

M. jubata

with 2 rows of papillae

with 3 – 6 rows of papillae

with 2 rows of papillae

with 3 – 4 rows of papillae

with 3 – 4 rows of papillae

pores separated by indistinct ribs

with prominent ribs

pores separated by indistinct ribs

Submarginal Anterior flange rib

Pores at the bottom of each mesh

Reticulum on the shield

8 –10

4 –10

smooth

smooth

smooth

smooth

smooth

single pore

single pore

occasionally a single pore

single pore

single pore

pores separated none by indistinct ribs

with a few irregularly scattered pores

smooth with few pores apically

26

18

13

50

36

thinly based 31 with a blunt peak/extension

with a small sharp peak

broadly based with a blunt peak

with a blunt 27 peak/extension

with a blunt peak

with a large pronounced peak

with a large pronounced peak

This paper, Asmund et al. (1982), Harris & Bradley (1957)

This paper

This paper, Asmund et al. (1982)

This paper

Nemcova et al. (2011)

Asmund et al. (1982), Harris & Bradley (1960)

Asmund et al. (1982)

This paper

No. of Relevant scales references used for GM analyses

with a helmet- 43 like dome extension

Posterior flange Rimmed pores Dome posteriorly on the shield

Table 1. A survey of diagnostic features on silicate structures in the Mallomonas pumilio group.

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Fig. 5. Shape changes of scales associated with extreme positions along first three relative warps.

The CVA documented significant separation among at least some of the a priori defined groups of scales (Wilk’s  = 0.0075, p < 0.0001). The first two canonical axes explained 77.3 % and 13.3 % of the shape variation, respectively (Fig. 3). Subsequent two-group tests illustrated that all the investigated groups were significantly different in shape (p < 0.001). The scale shape similarity between pairs of groups was compared using the values of Mahalanobis distance (MD). The smaller the value, the more similar in scale shape was the group pair. The most similar in shape of body scales were Mallomonas jubata × M. pumilio var. silvicola (MD = 7.15), morphotype A (M. solea-ferrea var. irregular) × morphotype C (M. pumilio var. dispersa; MD = 10.45) and M. pumilio var. munda × M. pumilio var. silvicola (MD = 12.57). A certain degree of overlap was observed among Mallomonas pumilio var. munda, M. pumilio var. silvicola and M. jubata scales on the CVA ordination diagram. On the other hand, the most dissimilar in shape was Mallomonas jubata × morphotype A (MD = 134.05). Along the first CV axis, taxa with wide flanges (Mallomonas solea-ferrea, morphotypes A, C and D) were separated from taxa with narrow flanges (M. jubata, M. pumilio var. munda, M. pumilio var. silvicola and morphotype B). Along the second CV axis, taxa were distributed based on the width of the body scales. However, no distinct line could be detected separating the wide-scaled (e.g. M. soleaferrea) and narrow-scaled (e.g. morphotype B) species. The figures of Mallomonas pumilio var. pumilio published by Asmund et al. (1982) comprise more than one morphotype. Their Fig. 14 (reprinted as Fig. 1B in this paper) corresponds to our morphotype B and their Fig. 15 (reprinted as Fig. 1C in this paper) resembles our morphotype C. The crucial task was to determine which of our morphotypes (A, B, C or D) may represent a type of Mallomonas pumilio var. pumilio, as described by Harris & Bradley (1957). The M. pumilio var. pumilio scale in Fig. 60 of Harris & Bradley (1960), reprinted as Figure 1 A in this paper (selected scale marked by the arrow), subsequently added to each of the eight groups, was repeatedly assigned to morphotype D, and the cross-validation procedure confirmed this assignment. Moreover, the scale morphology of this scale and the scales comprising morphotype D was congruent (compare Figs. 1A and 12E). Based on the above-described analyses we propose that the morphotype D should be identified with Mallomonas pumilio var. pumilio. Consequently, morphotypes A, B, and C should be described as separate taxa (see Table 1).

Fig. 6. A–M) Mallomonas jubata Nemcova, Kreidlova, Pusztai & Neustupa sp. nov. A–E) Body scales. 䉴 F–G) Collar scales with a well-developed, suboval dome. Note rimmed pores situated along the dorsal and proximal parts of the submarginal rib – arrows. H) A rear scale. I) An anterior part of the cell – collar scales with smooth, curved and attenuated bristles. J) A body scale. K) A whole scale-case. L) A collar scale. M) A whole scale-case. J–M) observed by scanning electron microscopy (SEM). Scale bar = 0.5 μm, unless stated otherwise.

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Taxonomy Mallomonas jubata Nemcova, Kreidlova, Pusztai & Neustupa sp. nov. (Figs 6 –7) Description: The collar scales (3.5 – 4.1 × 2.2 – 2.5 μm) are trapezoid-shaped (Figs 6F–G, L, 7 B) with a well-developed, suboval dome partly ornamented with circular meshes (Fig. 6G). Collar scales possess a typical extension bearing struts, sometimes forked at the end, fastened to the dome surface. The submarginal rib is well developed, continuously forming the edge of the extension. The flange is smooth, forming a prominent rounded protrusion on the dorsal edge. The proximal border encompasses the posterior and dorsal parts of the collar scale. The meshes of the reticulum are irregularly arranged and each encloses one to three pores. Four to ten rimmed pores are situated along the dorsal and proximal parts of the submarginal rib (Figs 6F–G, arrow). Body scales (3.1– 4.3 × 2.2 – 2.4 μm; Figs 6A–E) are hombic, domeless, and with a pronounced submarginal rib, which is attenuated on the lower anterior flange, i.e. the flange that is partly covered by an adjacent scale (e.g. Fig. 6B, arrow). One to two rows of body scales adjacent to the ring of collar scales are larger, the secondary reticulated layer may not be so distinct, and the meshes of the reticulum often enclose only a single depressed area (Figs 6C–E). Other body scales are smaller and bear a reticulation similar to the collar scales. An anterior flange is marked with short ribs connecting the lower part of the anterior submarginal rib with the rim of the basal plate. These ribs partly overreach to a posterior flange. There is a true pore (entered through the scale; visible on the side adjacent to the plasma membrane (Fig. 7A, arrow) placed anteriorly and several rimmed (5 –10) pores situated under/inside a posterior submarginal rib (Figs 6C, 7C). There is a fluent transition to the asymmetric rear scales (ca. 2.4 – 3.0 × 1.4 –1.9 μm; Figs 6H, 7C). The rearmost scales bear a diminutive spine. Bristles (5.4 –7.3 μm) are smooth, curved and attenuated (Figs 6I, 7A). Cells are ovoid (9.7– 15.0 × 5.1– 8.9 μm; Figs 6M, 7A). Cyst unknown. Distinguishing characters: 5 –10 rimmed pores, dome with a helmet extension; attenuated right submarginal rib on a body scale. Type: Figure 6 B. Type locality: an enclosed bay of Étang de Cazaux, Aquitaine, France (44° 26′ 27.34″ N; 1° 11′ 17.95″ W; pH 6.8; conductivity 236 S · cm–1), sampled on 4.2.2010. Etymology: The epithet means “having crest”, the dome extension resembles a Roman centurion’s helmet, surrounded by a transverse crest. Distribution: an unnamed pool close to Lac du Bousquet (Aquitaine, France; pH 7.1, conductivity 178 S · cm–1; 44° 30′ 1.90″ N, 0° 37′ 51.20″ W), a pool in riparian forest located between Étang de Yrieux and Étang de Beyres (Aquitaine, France; pH 7.2, conductivity 657 S · cm–1; N 43° 33.175′, W 1° 28.020′), Étang de Yrieux (Aquitaine, France; pH 7.6, conductivity 503 S · cm–1; N 43° 34.069′, W 1° 25.326′), Kunraticky Pond (Czech Republic, pH 6.2, conductivity 410 S · cm–1; temperature 3.1 °C; N 50° 44′ 59.1″, E 15° 6′ 3.6″), Podboranky Pond (Czech Republic, pH 4.6 (in the vicinity of Sphagnum sp.)–7.0; conductivity 182 (in the vicinity of Sphagnum sp.)– 326 S · cm–1; temperature 0.5 – 9.0 °C (throughout the year); N 50° 2′ 34.97″, E 13° 26′ 27.94″). Other records: Europe – Roijackers & Kessels (1981), Hartmann & Steinberg (1989). Mallomonas munda (Asmund, Cronberg & Dürrschmidt) Nemcova comb. nov. (Figs 3L–M) Mallomonas pumilio var. munda possesses a suite of features that clearly distinguished it from species of M. pumilio (M. pumilio var. pumilio and M. pumilio var. dispersa) which let us to raise

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Fig. 7. A–C. Mallomonas jubata Nemcova, Kreidlova, Pusztai & Neustupa sp. nov. A) A whole cell observed by scanning electron microscopy. Note a true pore, visible on the side adjacent to the plasma membrane – arrow. B) A part of the scale-case including collar and body scales. C) A group of body and rear scales. Scale bar = 0.5 μm.

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this variety to the rank of a separate species. The scales of M. munda are distinguishable from those of M. pumilio, in that they have pronounced submarginal rib delimiting narrow flanges. Moreover, the struts on the anterior flanges are rib like. On the other hand, the papillae arranged in rows oriented longitudinally with the scale border are typical for the anterior part of the M. pumilio. The reticulum of the shield of M. munda is roughly hexagonal, comprising mostly six pores at the bottom of each mesh, whereas in M. pumilio the meshes of the reticulum are not so regularly organized, comprising three to five pores and single pores (sometimes arranged in rows) in M. pumilio var. pumilio and M. pumilio var. dispersa, respectively. An additional distinctive feature is a large pointed peak of the dome in M. munda. The body scales of M. munda are also considerably larger compared to those of M. pumilio var. pumilio (morphotype D) and M. pumilio var. dispersa (morphotype C; see Fig. 4). Distinguishing characters: regularly shaped reticulum, anterior flange with prominent ribs, dome with a large pronounced peak. Type: Fig. 16 in Asmund et al. (1982). Basionym: Mallomonas pumilio var. munda Asmund, Cronberg & Dürrschmidt. Type locality: Als Island, Denmark, sampled on 13.3.1945. Etymology: The epithet “munda” refers to the beautifully elaborated reticulum of the shield (Asmund et al. 1982). Distribution: bipolar (Kristiansen 2002). Mallomonas silvicola (Harris & Bradley emend. Asmund, Cronberg & Dürrschmidt) Nemcova comb. nov. (Figs 8A–K) Similarly to the above species, we suppose that Mallomonas pumilio var. silvicola differs from M. pumilio taxa to such an extent that it should be distinguished as a separate species: M. silvicola. The scales of M. silvicola have a pronounced submarginal rib delimiting narrow flanges. Moreover, there are pores separated by struts on both the anterior and posterior flanges. A further important distinctive feature is the prominent pointed peak of the dome, which is even longer and more slender than in M. munda. The body scales of M. silvicola are also considerably larger compared to those of M. pumilio var. pumilio (morphotype D) and M. pumilio var. dispersa (morphotype C; see Fig. 4). Distinguishing characters: irregularly shaped reticulum, dome with a large pronounced peak. Type: the description of this species was based on four Figures (Harris & Bradley 1960; Figs 55, 59, 61, 62), which is in contradiction to the International Code of Botanical Nomenclature (art. 9.1). We selected their Fig. 55 to represent a lectotype. Basionym: Mallomonas pumilio var. silvicola Harris & Bradley. Type locality: not designated in the original description (Harris & Bradley 1960). Etymology: The epithet “silvicola” refers to the frequent occurrence in woodland ponds (Harris & Bradley 1960). Distribution: so far only known from Europe (Kristiansen 2002) Fig. 8. A–K. Mallomonas silvicola (Harris & Bradley emend. Asmund, Cronberg & Dürrschmidt) Nem- 䉴 cova comb. nov. A–C) Body scales. D–E) Rear scales. F) A collar scale with a bristle. G) A collar scale. H) A part of the scale-case – body and collar scales. I) A whole cell observed by scanning electron microscopy (SEM). J) A group of collar and body scales. K) A whole cell observed by SEM. Scale bar = 0.5 μm, unless stated otherwise.

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Mallomonas solea-ferrea var. irregularis Nemcova, Kreidlova, Pusztai & Neustupa var. nov. (morphotype A; Figs 9A–K) Description: The collar scales (2.9 – 3.3 × 1.8 – 2.1 μm; Figs 9H–J) of Mallomonas solea-ferrea var. irregularis are distinguishable from those of the nominal variety by the less distinctive shield pattern and more prominent (higher) peak of the dome. Both varieties have a very similar body scale morphology with no apparent submarginal rib and the shield covered with delicate papillae. The papillae are arranged in rows oriented longitudinally with the scale border at the anterior part of each scale (Figs 9B–C). Moreover, both M. solea-ferrea var. solea-ferrea and M. solea-ferrea var. irregularis possess smooth posterior flanges and two types of distinct pores; one is placed anteriorly (Fig. 9A, arrow) and the second, rimmed pore, is situated inside or under the posterior submarginal rib (Fig. 9A, arrowhead). The rear scales (1.2 – 2.2 × 0.9 –1.4 μm, Fig. 9D–F) are also similar to those of var. solea-ferrea. The body scales (2.4 – 3.0 × 1.6 – 2.0 μm; Figs 9A–C) of var. irregularis differ from the nominal variety in the more irregular pattern of the shield where the meshes of reticulum enclose various numbers of pores. Moreover the proximally formed depression (“window”) is considerably less extended compared to the horseshoe-shaped depression in var. solea-ferrea. Both varieties also differ significantly in the shape of body scales. The abovementioned features led us to separate these two morphotypes to the rank of varieties; however additional investigations using molecular techniques are necessary to evaluate the extent of genetic variation between these two taxa. Bristles (4.2 – 5.7 μm) are smooth, curved and attenuated (Figs 9I, 9K). Cells are ovoid (9.0 –11.8 × 4.0 – 5.9 μm; (Figs 9I, 9K). Cyst unknown. Distinguishing characters: irregularly shaped reticulum, smooth posterior flange, single rimmed pore posteriorly on the shield, less extended horseshoe-shaped depression compared to nominal variety. Type: Figure 9 A. Type locality: an artificial pool in the Natural Reserve Na Plachte near Hradec Kralove (Czech Republic, pH 5.6, conductivity 36 S · cm–1; N 50° 11′ 17.02″, E 15° 51′ 41.51″), sampled on 13.10.2010. Etymology: The epithet “irregularis” refers to the irregular organized reticulum on the shield of the body scale. Distribution: Podboranky Pond (Czech Republic, pH 6.3 –7.0; conductivity 257– 394 S · cm–1; temperature 7.3 – 23.2 °C (throughout the year); N 50° 2′ 34.97″, E 13° 26′ 27.94″). Mallomonas directa Nemcova, Kreidlova, Pusztai & Neustupa sp. nov. (morphotype B; Figs 10A–L). Description: The collar scales (3.3 – 3.7 × 2.2 – 2.7 μm; Figs 10E–F) have a broad base. An oval, broad-based dome is obtuse at the top, dorsally bearing a thin extension with a hinted peak. The flange is smooth. The shield is covered with regularly arranged reticulum. Each mesh consists of four to five pores and forms a depression relative to the scale’s surface. Dorsally, the groups of pores may incline to form rows (see Fig. 10E). The body scales (2.9 – 4.7 × 1.8 – 2.3 μm; Figs Fig. 9. A–K. Mallomonas solea-ferrea var. irregularis Nemcova, Kreidlova, Pusztai & Neustupa var. nov. 䉴 A–C) Body scales. A) Note two types of distinct pores: the anteriorly placed true pore (arrow) and the rimmed pore, situated under the posterior submarginal rib (arrowhead). D–E) Rear scales. F) Rearmost scales with a short spine. G) A whole cell. H) Collar scales. I) A whole cell observed by SEM. J) Collar scales. K) A whole cell observed by SEM. Scale bar = 0.5 μm, unless stated otherwise.

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10A–D) are dimorphic. Two rows of pores are separated by distinct struts in the longer central scales, while groups of pores are surrounded by the less prominent struts (Figs 10C–D). On the other hand, the body scales placed apically and posteriorly on the cell are considerably wider, bearing reticulation similar to the collar scales. The submarginal rib is distinct, but is however inflated and confluent with the flanges. The row of minute papillae is visible on an anterior flange; the posterior flange is smooth. The rear scales (1.5 – 2.6 × 1.0 –1.6 μm; Figs 10G–I) are asymmetric. The reticulation of the shield is more or less confluent at the rearmost scales (Fig. 10K). No spine was observed. Bristles (ca. 5 – 6 μm) are smooth, slightly curved. Cells are ovoid (approx. 11–16 × 4 – 9 μm; based solely on the measurement of two intact scalecases). The cyst is ellipsoidal to spherical with the pore surrounded by a flaring collar. The surface of the cyst is covered by minute papillae (see Fig. 12 in Asmund et al. 1982: here Mallomonas directa was determined as M. pumilio var. pumilio). Distinguishing characters: dimorphic body scales, regularly shaped reticulum, in central scales forming rows, broadly based dome with a blunt peak. Type: Figure 10 E. Type locality: an enclosed bay of Étang de Cazaux, Aquitaine, France (44° 26′ 27.34″ N; 1° 11′ 17.95″ W; pH 6.8; conductivity 236 S · cm–1), sampled on 4.2.2010. Etymology: The epithet “directa” refers to the direct rows of pores in the centrally located body scales. Distribution: Kunraticky Pond (Czech Republic, pH 6.2, conductivity 410 S · cm–1; temperature 3.1 °C; N 50° 44′ 59.1″, E 15° 6′ 3.6″), Pekelsky Pond (Czech Republic, pH 6.0, conductivity 203 S · cm–1; N 50° 44′ 21.0″, E 15° 6′ 38.4″), a small unnamed pool (Czech Republic, pH 5.9, conductivity 230 S · cm–1; N 50° 44′ 36.3″, E 15° 6′ 18.8″), an artificial pool (Czech Republic, pH 5.6, conductivity 36 S · cm–1; N 50° 11′ 17.02″, E 15° 51′ 41.51″), and a pool in a riparian forest (Aquitaine, France; pH 6.2, conductivity 521 S · cm–1; temperature 11.8 °C; N 43° 30.464′, W 1° 15.565′) Other previous records: Europe – Roijackers & Kessels (1981), South America – Dürrschmidt (1980), Dürrschmidt (1982), Asmund et al. (1982). Mallomonas pumilio var. dispersa Nemcova, Kreidlova, Pusztai & Neustupa var. nov. (morphotype C; Figs11 A–K) Description: The collar scales (2.8 – 3.4 × 1.7– 2.3 μm; Figs 11A, C, D) bear a relatively small, sideward-positioned dome provided with a small sharp peak. The submarginal rib is well developed at the dorsal edge and ventrally, just beneath the dome. The flange is smooth. The shield is covered by regularly spaced rows of delicate papillae, which may be absent on some parts of the scale (Fig 11A). Single pores are dispersed more often on the posterior part of the scale, and in addition a row of pores lines the dorsal submarginal rib. The body scales (2.6 – 3.3 × 1.7– 2.2 μm; Figs 11F, G, I, J) are almost flat, possessing an indistinct submarginal rib; however an inflated submarginal rib confluent with flanges is typical for rear scales (see Figs 11E–F). The shield of Fig. 10. A–L. Mallomonas directa Nemcova, Kreidlova, Pusztai & Neustupa sp. nov. A–D) Dimorpic body 䉴 scales. A–B) Body scales placed apically and posteriorly on the cell. C–D) Larger central body scales with pores organized to rows. E–F) Collar scales with an oval, broad-based dome. G–I) Rear scales. J) An anterior part of the cell. K) Scales from the posterior part of the cell. L) A whole cell observed by SEM. Scale bar = 0.5 μm, unless stated otherwise.

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the body scales is ornamented by regularly spaced rows of delicate papillae and more or less regularly organized pores, which may sometimes form rows (Fig. 11G). Several pores are joined up to form one to three groups posteriorly on the shield. The anterior flange is covered by rows of delicate papillae, and the posterior flange is smooth. The rear scales (1.5 –1.7 × 0.9 –1.1 μm) are asymmetric, the rearmost scales possess a thin basal plate with a few pores and are distally ended by a diminutive spine (ca. 0.2 μm). Bristles (4.4 – 6.2 μm) are smooth, slightly curved (Figs 11H, 11K). Cells are ovoid (8.0 –11.5 × 3.0 – 4.6 μm). Cyst unknown. Distinguishing characters: shield with dispersed single pores (sometimes arranged in rows), anterior flange covered with 3 – 6 rows of papillae, dome with a small sharp peak. Type: Figure 11 I. Type locality: Pekelsky Pond (Czech Republic, pH 6.0, conductivity 203 S · cm–1; N 50° 44′ 21.0″, E 15° 6′ 38.4″), sampled on 4.4.2011. Etymology: The epithet “dispersa” refers to the pores dispersed on the shield. Distribution: a small unnamed pool (Czech Republic, pH 5.9, conductivity 230 S · cm–1; N 50° 44′ 36.3″, E 15° 6′ 18.8″), Podboranky Pond (Czech Republic, pH4.6; conductivity 182; temperature 7.8 °C; N 50° 2′ 34.97″, E 13° 26′ 27.94″), and an enclosed bay of Étang de Cazaux, (France, pH 6.8; conductivity 236 S · cm–1; N 44° 26′ 27.34″, W 1° 11′ 17.95″). Other previous records: Europe – Asmund et al. (1982), North and Central America – Jacobsen (1985) Mallomonas pumilio var. pumilio Harris & Bradley emend. Nemcova et al. (morphotype D; Figs 12A–I) Description: The collar scales (2.9 – 3.5 × 1.9 – 2.3 μm) possesses a smooth oval dome with relatively thin base and a blunt beak (Figs 12C, 12I). In body scales (2.8 – 3.3 × 1.6 – 2.2 μm; Figs 12E, F, H) the anterior submarginal rib is well developed, forming a frame in which two adjacent scales are fixed. The posterior submarginal rib is less prominent; it is not clearly delimited against the shield and flange. The anterior flange bears longitudinal rows of papillae (mostly two); the posterior flange is smooth. The shield is covered by regularly spaced groups of pores. Each group of pores (3 – 5) forms a slight depression. Reticulation is proximally confluent, forming a window. The rimmed pore is situated inside or under the posterior submarginal rib (it is not visible on SEM micrographs) just behind the window. The pattern of reticulation is similar in all body scales. The rearmost scales (1.2 – 2.6 × 0.9 –1.6 μm) possess spines (up to 1.4 μm) and may lack reticulation (Fig. 12D). Bristles are smooth, curved and attenuated. Distinguishing characters: regularly shaped reticulum, 3 – 5 pores within a mesh, anterior flange with rows of papillae, thinly based dome with a bluntly cut extension. Epitype: Figure 12 E (lectotype: Fig. 4 in Harris & Bradley 1957; selected by Asmund et al. 1982; the lectotype, represented by carbon replica, cannot be critically identified for purposes of the precise application of the name of a taxon). Fig. 11. A–K. Mallomonas pumilio var. dispersa Nemcova, Kreidlova, Pusztai & Neustupa var. nov. A) A 䉴 collar scale with a small dome provided with a small sharp peak. B) A body scale with group of scales from the posterior part of the cell. C) A collar scale. D) Collar scales and a body scale. E) Whole cell armor. F) A body scale with a rearmost scale. G) A body scale observed by SEM. H) A scale-case observed by SEM. I–J) Body scales. K) A scale case observed by SEM. Scale bar = 0.5 μm, unless stated otherwise.

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Previous records: Europe – Harris & Bradley (1960), Hansen & Kristiansen (1997), Kristiansen & Vigna (1996), Hartmann & Steinberg (1989), Němcová et al. (2003), North and Central America – Wujek & Bland (1991), Nicholls (1988), South America – Siver & Vigna (1997).

Discussion Even though implementation of electron microscopy allowed detailed investigation of scale morphology, there are still species within species-complexes that are difficult to discern/determine. The taxa of the Mallomonas pumilio group represent the dwarfs of the genus. The cells, reaching a length of around 12 μm, may slip unnoticed through the mesh of a plankton net (plankton nets do not usually have a mesh size smaller than 20 μm). Therefore, records of these species are probably underestimated. Ideally, a combined molecular and morphometric approach would be used for Mallomonas pumilio group taxonomy, with a priori groups defined on the basis of molecular data (Verbruggen et al. 2005, Nemjová et al. 2011). However, it is extremely difficult to isolate and cultivate such small species, and no molecular data has so far been available. Therefore, we attempted to provide taxonomy analysis based on shape data combined with ultrastructural information. In this context, we defined a priori groups on the basis of detailed scale and scale-case morphology and, subsequently, we used the multivariate statistical procedures to test differences in shape between these groups. However, we are aware that, without molecular phylogenetic studies, cryptic diversity of species with identical scale morphology cannot be discerned. Along the first CV axis, taxa possessing small scales with wide flanges (Mallomonas soleaferrea, M. solea-ferrea var. irregularis, M. pumilio var. dispersa and M. pumilio var. pumilio) were separated from large-scaled taxa with narrow flanges (M. jubata, M. munda, M. silvicola and M. directa). Dimorphic body scales of M. directa comprised both wide-flanged (centrally located) scales and narrow-flanged scales. However, both types of scales were significantly larger than other wide-flanged taxa. All the investigated groups of scales were significantly different in shape, even though a certain degree of overlap was observed (see Fig. 3). Although we used a statistically powered GM approach, the a priori definition of the groups, representing distinctive taxa, may include a certain degree of subjectivity. Molecular data are needed to evaluate whether these taxonomic units should be treated as independent species. However, considering the present state of knowledge, we believe that treating M. jubata, M. munda, M. silvicola and M. directa as distinct species, while treating M. solea-ferrea var. irregularis, M. pumilio var. dispersa as varieties, is the best working hypothesis. Landmark-based geometric morphometric approaches combined with morphology have been successfully used to solve taxonomic clues in phycology. Geometric morphometric data were used together with valve morphology to clarify the identity of a centric diatom morph, which showed intermediate morphology between that of typical specimens of Cyclotella meneghiniana Kützing and of Cyclotella scaldensis Muylaert & Sabbe (Beszteri et al. 2005). Since this early study, the sliding landmarks (semilandmarks) have been introduced and widely implemented. Semilandmarks, which slit along the outline curve, help to capture the shape of the structure when an insufficient number of biologically meaningful points (landmarks) can be identified (Bookstein 1997). In Mallomonas taxonomy, landmark-based GM methods (using sliding land䉳 Fig. 12. A–I. Mallomonas pumilio var. pumilio Harris & Bradley emend. Nemcova, Kreidlova, Pusztai & Neustupa. A) Whole cell armor. B) A posterior part of the cell. C) Collar scales with smooth oval dome. D) Rearmost scales possessing spine. E) A body scale. F) A body scale observed by SEM. G) A whole cell observed by SEM. H) Body scales. I) A whole cell observed by SEM. Scale bar = 0.5 μm, unless stated otherwise.

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mark algorithm) were also employed to distinguish between scales belonging to the two distinct varieties: M. striata var. striata and M. striata var. serrata. A priori groups were defined on the basis of the morphology of bristles (Neustupa & Němcová 2007). Mallomonas striata var. striata possesses smooth and M. striata var. serrata serrated bristles (Harris & Bradley 1960). There is only scarce information on the ecological preferences of individual taxa comprising the Mallomonas pumilio group. Generally, they inhabit neutral to slightly alkaline environments with a low to medium conductivity. Interestingly, Mallomonas pumilio complex taxa seem to be much more common in Europe than in North America. Neither species of this complex has been reported during extensive silica-scaled chrysophyte research conducted along the east coast of North America between 1991 and 2006 (Siver & Lott 2012). However there are some records from other studies (Wujek & Bland 1991, Nicholls 1988, Jacobsen 1985). There is also a well documented record of Mallomonas directa from South America (Chile, Asmund et al. 1982). The method presented in this study, which unites geometric morphometric data and detailed information on scale and scale-case morphology, may help to elucidate taxonomic issues within the Mallomonas pumilio group, for which no molecular data are available. The application of geometric morphometric methods opens a variety of perspectives for the study of both the taxonomy and ecology of silica-scaled chrysophytes.

Acknowledgments John Wiley and Sons Inc., publishing company, is acknowledged for permission to copy Figures 1 A–C (Licence Agreement 3006381258544). This work was supported by the Institutional Funds of the Charles University in Prague.

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