Morphological Evolution of Stratiotes through the ... - GeoScienceWorld

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RESEARCH REPORT

Morphological Evolution of Stratiotes through the Paleogene in England: An Example of Microevolution in Flowering Plants NICHOLAS P. SILLE Geology Department, Royal Holloway University of London, Egham, Surrey, TW20 OEX, UK; and Palaeontology Department, Natural History Museum, Cromwell Road, London, SW7 5BD, UK, E-mail: [email protected] MARGARET E. COLLINSON, MICHAL KUCERA* Geology Department, Royal Holloway University of London, Egham, Surrey, TW20 OEX, UK JERRY J. HOOKER Palaeontology Department, Natural History Museum, Cromwell Road, London, SW7 5BD, UK

PALAIOS, 2006, V. 21, p. 272–288

DOI 10.2110/palo.2005.P05-21e

Six populations of seeds of the aquatic monocotyledon Stratiotes (Hydrocharitaceae) from the Paleogene of England have been studied to assess morphological evolution through the Eocene–Oligocene transition. Morphometric methodologies (including eigenshape analysis) have been used to quantify evolution within the genus and compare results to previous qualitative studies. Previously hypothesized broad evolutionary trends of increasing size and more elongate shape are found to be mainly correct, but, in places, can be elucidated further. The results of this study indicate a single evolving lineage in the Paleogene of southern England with an increase in seed size and keel width in late Eocene specimens, followed by a reversal of this trend in the early Oligocene. Two Miocene populations from continental Europe are shown to be morphologically distinct from those of the English Paleogene. Changes in overall shape of the seed are shown to be controlled dominantly by the relative size of the keel structure, rather than the seed body. Comparisons show that the microevolutionary trend of Stratiotes across the Eocene–Oligocene transition differs from that of the charophyte, Harrisichara. This may suggest that factors other than climatic change, such as animal/plant interactions, played a role in evolution of Stratiotes seeds. Type and figured material of named Paleogene species was added passively to the dataset, and results suggest that taxonomic splitting may have led to previous evolutionary hypotheses of multiple clades, which is not supported by this study.

INTRODUCTION The Late Paleogene is marked by a series of major Cenozoic global climatic events that strongly affected patterns of distribution in the world’s biota. The Paleocene– Eocene transition is an abrupt episode of global warming, characterized by a temperature increase of 5–68C in oce*Current address: Institut fu¨r Geowissenschaften, Universita¨t Tu¨bingen, Sigwartstrasse 10, 72076 Tu¨bingen, Germany

anic bottom waters (Kennet and Stott, 1991) and 88C in high-latitude surface waters. Following the Eocene climatic optimum, the middle Eocene onwards is characterized by a gradual cooling (Zachos et al., 2001) followed by the Eocene–Oligocene transition, which marks the change from greenhouse to icehouse conditions. This global cooling culminated in the build up of the Antarctic ice sheet (Oi-1 glaciation at 33.5 Ma) 200 kyrs after the Eocene–Oligocene boundary. These changes had major effects on global biotas (Prothero and Berggren, 1992; Meng and McKenna, 1998; Hooker et al, 2004; Prothero et al., 2004). The strata of the Hampshire Basin, southern England, provide a unique opportunity to study how the continental realm responded to the global changes of the Late Paleogene (Collinson et al., 1993; Hooker in Prothero and Berggren, 1992; Sille et al., 2004; Hooker et al., 2004). Biotic changes can be traced in both faunal and floral records, with several mammal faunal turnover events observed both before and after the Eocene–Oligocene boundary (Hooker et al., 1995, 2004). The response of late Paleogene floras of the Hampshire Basin includes an increase in temperate elements (Collinson in Prothero and Berggren, 1992). The overall vegetation shifted from a diverse forest of tropical aspect in the Middle Eocene to an environment dominated by swamps and marshes with patches of woodland of partly tropical aspect by the Late Eocene. No major vegetational change has been recognized between the latest Eocene and the earliest Oligocene (Collinson, 1983; Collinson et al., 1993; Hooker et al., 2004). Morphological evolution has been observed in charophytes (green algae) in the latest Eocene but, as with the vegetation as a whole, not specifically across the Eocene–Oligocene transition (Sille et al., 2004). Although biotic change across these intervals has been documented qualitatively within the fauna and flora, quantitative studies of the plant record have been few. There has been only a single study on charophycean algae (Sille et al., 2004), and, to date, no detailed morphometric work on angiosperm seeds. The present study documents a morphometric analysis of seeds of the aquatic monocot genus Stratiotes (Hydrocharitaceae) through the Paleogene, with particular attention to the Eocene–Oligocene transition in southern England. Results are used to ad-

MORPHOLOGICAL EVOLUTION OF STRATIOTES

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TABLE 1—List of samples; note that sample names (except BM1) do not refer to the same samples as used in Sille et al. (2004). Sample name MM2 MM1 HM2 HM1 BM1 BL1 HH1 RD1

Locality

Country

Age

Numbers of specimens

Belchato´w North Bohemian Basin Bouldnor Cliff Bouldnor Cliff Thorness Bay Gurnard Hordle Cliff Felpham

Poland Czech Republic UK UK UK UK UK UK

Early Miocene Early Miocene Early Oligocene Early Oligocene Early Oligocene Late Eocene Late Eocene Early Eocene

65 83 43 31 34 42 37 25

dress the nature and timing of any morphological changes observed within the genus. Comparisons are made with previous evolutionary hypotheses in the light of the new quantitative data and new interpretations of evolution within the genus Stratiotes are made. Type and figured material from southern England are then superimposed onto the analysis. This tests the current taxonomic scheme in relation to the populations that have been sampled during this study and assesses whether this taxonomic scheme may have influenced previous interpretations of evolution within the genus. The manifestation of morphological shifts in the seeds and the functional significance of these changes are discussed, and observed patterns of evolution are compared and contrasted to previously documented floral and faunal change. MATERIAL AND SAMPLING HORIZONS This analysis is based on the study of populations of mature seeds from six English Paleogene samples, with two continental European samples of Miocene age included for comparison (Table 1). Seeds of Stratiotes were selected for this study for several reasons. They are character rich (both internally and externally; Fig. 1), maturity can be recognized easily, and they are deposited in the aquatic environment in which they grew and, therefore, rarely are transported (this can be shown by the quality of the pres-

FIGURE 1—Stratiotes seed morphology. (A) Line drawing, internal view of a Stratiotes seed valve with anatomical nomenclature. (B) SEM image of internal view of a Stratiotes seed valve showing the basic measurements used in the analysis.

ervation observed and the associated biota). The genus also is known to exhibit morphological variation through the Paleogene, based on previous, mainly qualitative, studies (Chandler, 1923; Holy´ and Bu˚zˇek, 1965; Mai, 1985; Collinson, 1990: p. 50–52). Furthermore, the relative abundance of seeds within the horizons provides potential for a quantitative morphological analysis. Six Stratiotes-bearing horizons from five different localities in southern England were included in this study (Figs. 2, 3; Table 1). The samples range in age from the Paleocene/Eocene transition to the earliest Oligocene. The oldest sample used in this study is the oldest fossil record of Stratiotes. The sample was collected from clays above the Felpham Lignite Bed (Reading Formation; see Bone, 1986). Recent studies on the Paleocene–Eocene boundary (Collinson and Cleal, 2001; Collinson et al., 2003) in southern England, and correlation of paleobotanical assemblages from this time period, have suggested a date for the Felpham material of ;100kyr younger than the carbon-isotope excursion marking the Paleocene–Eocene boundary (Collinson and Cleal, 2001; Collinson et al., 2003), making this Stratiotes sample earliest Eocene in age. The upper five samples are from the Solent Group (late Eocene to earliest Oligocene in age), and were collected from sections with observable superposition of strata (Table 1; Fig. 3). Therefore, the age could be constrained fairly easily (Insole and Daley, 1985; Hooker et al. 2004; Fig. 3). Sample locations are as follows: HM2—Gray mud, 3.9 m above level HM5 of Sille et al. (2004); HM1—Gray mud with Viviparus, 3 m below Nematura Bed, at Bouldnor Cliff (see Sille et al. 2004, fig. A1 for log); BM1—Gray mud with pulmonate gastropods, the lower part of Daley (1973) bed XVII, 4.5 m above greenish white band, in cliff below Thorness Wood, in Thorness Bay; BL1—Green mud, top 10 cm

FIGURE 2—Location map of southern England showing the sites from where samples were collected.

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FIGURE 3—Stratigraphic position of samples with correlation of important global and local events (Insole and Daley, 1985; Stuchlik et al., 1990; Berggren et al., 1995: Sille et al., 2004; Hooker et al., 2004); Fm.5Formation; Mbr.5Member.

of Daley and Edwards (1990), bed 6c from Gurnard; and HH1—Chara bed (also the base of bed 28) of Edwards and Daley (1997), at Hordle Cliff, Hampshire. Miocene specimens were included from Poland and the Czech Republic. The Polish material is from the early Miocene of the Belchatow mine (Stuchlik et al. 1990; Worobiec, 2003). A sample of 72 unnumbered specimens was loaned from the Polish Academy of Sciences. The Czech seeds are from the North Bohemian Basin (from Zapoˇ ernovice, Moraveves, Chomutovsko, Ejovice, and tocky, C Vrsany; National Museum Prague numbers 3089, 3090, 3091, 3096, 3099, 4003, 4004, and 4007) and are mostly from the main seam complex called the Holesice Member of the Most Formation. The base of the seam is dated to Neogene mammal zone MN 3, Burdigalian, Early Miocene (J. Kvacˇek, pers. comm., 2004). In all, eight samples were studied, each between 25 and 83 specimens, with 360 specimens included in the analysis (Table 1). METHODS: SAMPLE PROCESSING AND DATA COLLECTION Bulk sediment from southern England was disaggregated using hydrogen peroxide and then sieved through a 1mm mesh. All specimens of Stratiotes were picked from the sieved residues and cleaned in hydrofluoric acid. The recognition of Stratiotes was based on the seed characters used by Chandler (1923). The specimens were re-picked and only complete, mature valves were selected for further study (germination of the seeds proves seed maturity, therefore the analysis is not skewed by the introduction of immature specimens). No specific determinations were made on the seeds. This approach was taken to gain an appreciation of the overall variation within populations of the genus without introducing a-priori bias caused by specific determinations. This method has been used previously in paleobotany and wider fields of paleontology (Sille et al., 2004; Girard et al., 2004).

The genus Stratiotes, with the single modern species S. aloides L. (water soldier), is an extant, fully aquatic angiosperm, and a member of the monocotyledon family Hydrocharitaceae. It is aquatic, freshwater, and rooted to a freefloating plant, which is unique in the sense that it sinks in the autumn and then floats to the surface during spring, and is partially emergent during the summer months. Although it is partially emergent and bottom rooted in summer, Stratiotes cannot grow as a terrestrial plant and therefore is not found in temporary water. It mainly is found in water 2–5 meters deep and confined to nutrientrich, loose muddy substrates (Cook and Urmi-Ko¨nig, 1983). The fossil record of Stratiotes is restricted to Europe and extends back to the Paleocene–Eocene boundary (Collinson in Bone, 1986). The oldest fossil example known is included in this analysis. The majority of the fossils are entire seeds or single valves of germinated seeds. However, some leaf fragments (margin teeth) also have been found (Collinson, 1983; Collinson et al., 1993). The history of paleobotanical study of Stratiotes has been complex (see Chandler, 1923 p. 117–118, for discussion of study pre-1923), and a number of species have been named from the fossil record. Owing to the lack of the whole plant, which is used for modern descriptions, fossil species are described based on the gross morphology and cell structure of their seeds (Chandler, 1923 and references within; Palamarev, 1979). Fossil seeds of the genus Stratiotes can be recognized based on a number of seed characters defined by Chandler (1923). The seeds are oblong, generally narrow, and often are hooked at the base (Fig. 1). The seeds are distinct in the sense that they possess a keel on the dorsal edge that contains the raphe. In some cases, the raphe traverses the length of the keel and exits near the base of the specimen (see below for fuller discussion of raphe). The ventral side of the base of the seed possesses a collar that is penetrated by the micropyle (which narrows markedly towards the exterior), and the presence of the collar produces the hooked nature of the seed. The external surface of the seeds is characterized by a number of longitudinal ridges that vary in both size and shape. Each of the specimens of Stratiotes was oriented in an internal lateral view (Fig. 1) for study. A SONY DXC-390P 3CCD color video camera connected to a Leica MZ 125 light microscope was used to obtain images. The outlines of the specimens were digitized using NIH-Image (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/). Five characters were measured using image-analysis software, and shape was studied using eigenshape analysis: length, length/width ratio, keel width, seed-edge width, and seed-body length (Fig. 1). Several other seed characters previously used in species descriptions that were excluded include features of the raphe, cellular detail of the keel, presence of mucilaginous cells on the seed coat, and the pattern of external ridges. The position of exit of the raphe on the external seed margin previously was used as a specific discriminator by Chandler (1923, 1957, 1961). After studying the specimens, however, it was recognized that, in many cases, it was extremely difficult to obtain an accurate measurement of the exact position of the exit of the raphe, especially when the raphe runs down the dorsal edge of the


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keel, leaving the exact exit point unclear. Cell detail on the keel of the valves is a character used by Chandler (1923). To include this in the present analysis is not feasible for a number of reasons; one is that the clarity of cell outlines varies between specimens. The reasons for this are not well understood, but the result is that cell characters could not be documented for all specimens. In addition, the detail, where visible, has been found to be variable in nature along the length of the seed, and the variable course of the raphe makes it difficult to study comparable positions on all seeds. The presence of spirally thickened mucilaginous cells on the outer surface of the seed coat is used in this paper as a measure of seed preservation. Previous studies have shown that the presence of mucilaginous cells on the external coat of the seeds is a good indicator for lack of chemical corrosion of the seed (Hooker et al., 1995). Hooker et al. (1995) also showed that where the mucilaginous cells are missing, the seeds had undergone substantial chemical corrosion, which also may affect the shape and size of the seeds. In the assemblages studied herein, the presence of the mucilaginous cells has been used as an indication that the seeds have not suffered from loss of tissues, which might have altered their shape and size. Chandler (1923) used aspects of the external ridges to distinguish species. However, the authors judge that the ridges are more variable than indicated by Chandler and more complicated (making consistent quantification extremely difficult). Furthermore, the outer spirally thickened cells partially obscure the ridges in the well-preserved specimens that have been selected for their unaltered size and shape to be most appropriate for this study. Eigenshape Analysis Analysis of shape (as used in Sille et al., 2004) was conducted using eigenshape analysis (Lohmann, 1983). Coordinate data were collected on internal lateral views of digital images of the Stratiotes valves using NIH-Image software. The outlines were interpolated into a series of evenly spaced points and converted into the w-format shape function by calculating angular differences between consecutive points. These were transformed to the w* format by subtracting the net angular bend attributable to a circle. Eigenshape analysis as used in this paper involves a singular value decomposition of the covariance matrix among individual w*-shape functions (MacLeod and Rose, 1993). Transformations of the original coordinates of the outline points into the w* format were performed using the Eshape package program written by Norman MacLeod, freely available on the Internet (http://www.nhm.ac.uk/ hostedpsites/paleonet/). MATLABt routines written by MK were used for the purpose of further analysis, including the singular value decomposition and the modeling of shape variability along the eigenshapes (Kucera, 1997). RESULTS Eigenshape Results Two distinct eigenshape analyses were carried out on the mature Stratiotes valves to quantify shape variation within the genus. The first of these was a standard outline

FIGURE 4—Eigenshape analyses. (A) Results of the two eigenshape analyses with explanation of the four derived characters. (B) Graphical representation of the outline used for the outline eigenshape analysis (the homologous point used was the center of the micropyle on the external surface of the valve). (C) Graphical representation of the outline used for the seed-coat outline eigenshape analysis (the homologous point used was the exit of the micropyle on the external surface on the ventral side of the specimen). See Figure 8E for scaled figure.

eigenshape analysis (using 50 equally spaced coordinate points) using the center of the micropyle as the homologous point (Fig. 4B). This analysis derived three distinct shape characters (encompassing 88.8% of the total shape variation) for further analysis (Fig. 4A). The first eigenshape shows differences in overall outlines of the specimens, varying from elongate to more rounded forms. The second eigenshape shows the relative location of the micropyle, from exiting the valve at the center of the base of the seed to exiting on the ventral margin of the seed. The third eigenshape documents the size of the collar, varying

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FIGURE 5—Mean values (with 95% confidence intervals) of measured morphological characters showing varied patterns in relation to stratigraphic order.

from forms with little or no collar to forms with large, welldeveloped collars. The second eigenshape analysis (using 100 equally spaced coordinate points) using the ventral side of the exit of the micropyle as the homologous point, traced both the internal and external seed-coat outline (Fig. 4C). This analysis derived one shape character (encompassing 97.4% of the total shape variation) showing variations in the seed-coat outline. Single Variable Results To illustrate patterns within the data better, mean values of each level were calculated (Fig. 5). A number of trends can be observed from these mean character values. In the Paleogene of the Hampshire Basin, seeds from levels RD1 and HH1 are small (Fig. 5A), then there is an increase in size in BL1 and BM1. Seeds in levels HM1 and HM2 are smaller again in size and similar to the oldest levels. There is then a size increase to the Miocene levels, with those of MM2 being the largest. Length/width ratio shows a very different trend to that of length (Fig. 5B). RD1 has a length/width ratio of approximately 2. There is then a marked decrease in levels HH1, BL1, and BM1. The upper two levels from the Hampshire Basin, and the two Miocene levels have an increased value similar to that of RD1. Keel width/width ratio (keel-width ratio; Fig. 5C) shows almost the exact opposite of length/width ratio, with HH1, BL1, and BM1 exhibiting high values (relative-

ly large keels) and the rest of the levels having lower values (relatively smaller keels). More negative values of 1st eigenshape represent more elongate specimens where less negative values represent specimens more rounded in shape (Fig. 4A). Figure 5D shows that BL1 is the least elongated followed closely by HH1 and BM1; the remaining levels are more elongated in shape. The length/width ratio of the central cavity, representing the space originally containing the embryo and storage tissue (here termed seed body), also was calculated. The results showed that there was very little change through time (all values are within the margin of error). This indicates that any shape changes of the overall seed are not caused by the seed body, but by parts of the seed coat. Multivariate Results An R-mode principal component analysis (PCA) was carried out on all nine variables to understand patterns within the data better. The first principal component (PC1; 32.4% of the total variance) is dominated by length/ width ratio, keel-width ratio, 1st eigenshape, and seed-coat outline (Table 2), showing that the variation within PC1 is dominated by shape variation. The second principal component (PC2; 20.7% of the total variance) is dominated by length, and to a lesser extent, by 1st eigenshape. The third principal component (PC3; 13.2% of the total variance)


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TABLE 2—Principal component loadings and variation explained by the first three principal components of Stratiotes morphometric analysis.

Length Length/Width ratio Seed Body Length/Length ratio Keel Width/Width ratio Edge Width/Width ratio 1st Eigenshape 2nd Eigenshape 3rd Eigenshape Seed-coat outline Variation explained (%)

1st PC

2nd PC

3rd PC

0.038 20.847 20.339 0.753 0.249 0.699 20.196 20.637 0.724 32.4

0.835 0.306 20.610 0.093 0.366 20.536 20.487 20.188 0.026 20.7

20.141 0.322 0.026 0.093 0.557 20.241 0.708 20.147 0.403 13.2

scores are dominated by 2nd eigenshape and edge-width ratio. The mean level values of PC1 (Fig. 6A) show an increase from RD1 to HH1; these values then remain constant through levels BL1 and BM1. The values then decrease markedly to level HM1. They then stay relatively constant. The graph in Figure 6A distinguishes levels HH1, BL1, and BM1, for possessing relatively large keels, a low length/width ratio, and having a more rounded overall shape. The remaining five levels have relatively small keels, a higher length/width ratio, and a more elongate overall shape. The mean values of PC2 (reflecting changes in overall size of the seeds; Fig. 6B) show an increase from level HH1 to BM1, then a decrease in levels HM1 and HM2. Following this is a large increase to the Miocene samples MM1 and MM2. PC3 (Fig. 6C) shows a different pattern again, with RD1 exhibiting the highest value and a decrease through levels HH1 and BL1. There is a slight increase in BM1, then a larger increase in HM1 and HM2. The Miocene levels possess slightly lower values. When PC1 is plotted against PC2 (Fig. 7A), the individual specimens from each level can be seen to show a large amount of overlap (see range polygons for selected levels, Fig. 7A). When the mean values (with 95% confidence intervals) for each level are calculated (Fig. 7B), the data are easier to interpret in terms of changes in size and shape of the seeds. The plot (Fig. 7B with representative seed outlines; also see Fig. 8) shows that the means for each level are distinctly different from each other even though the spreads of individual specimens overlap. This shows that the analysis of populations of specimens is important to reveal the extent of variation, and to give reliable mean values of the population as a whole for comparison with other populations. Normality of Distribution Goodness-of-fit tests were carried out on all the data to test for normality of distribution (see Fig. 9 for histogram stacks). A lack of bimodality in the data would suggest that there is no evidence of co-occurrence of two or more species at any of the sampled levels. The data were assumed to be parametric, and chi-squared tests were carried out on all the variables in every level (with Yates’ continuity correction where necessary). Of the 96 chi-squared tests, eight rejected the hypothesis of normality at a 5%

FIGURE 6—Mean values (with 95% confidence intervals) of the first three principal components showing varied patterns in relation to stratigraphic order.

level. Seven of these occurred in the levels with the smallest number of specimens, and all involved using Yates’ continuity correction (in samples RD1 and HM1). The other test that rejected the hypothesis of normality was the PC1 data of level MM2. Out of the remaining 88 tests, nine gave results within 1.0 of the critical value. These 17 tests were recalculated using the Kolmogorov-Smirnov goodness-of-fit test as a means of checking the results for nonparametric data. In all 17 cases, there was a 95% confidence that they could not be distinguished from a normal distribution. Therefore, the results of the two statistical analyses indicate that there is no bimodality in any of the variables throughout all the levels. Comparisons with Type and Previously Figured Specimens When the analysis of the current data set was complete, type and figured specimens of the five species of Stratiotes

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FIGURE 7—Principal components results. (A) Principal components plot showing all specimens, with range polygons showing the extent of variation within selected levels. (B) Mean values for levels with 95% confidence intervals and representative outline drawings to scale.

(S. headonensis Chandler, 1923; S. hantonensis Chandler, 1960; S. websteri (Cuvier and Brongniart, 1822) Zinndorf, 1901; S. neglectus Chandler, 1923; and S. acuticostatus Chandler, 1923) previously reported in southern England (Table 3) were imaged and measured in the same way as the seeds sampled in this study. These data were added passively to the eigenshape analyses. Then, the complete sets of data on the type and figured specimens were added passively to a principal-components analysis of the six levels from southern England (Fig. 10). This was carried out to test both the application of the current taxonomy of the species in relation to complete populations of seeds, and to

assess if taxonomic constraints may have affected evolutionary interpretations in previous studies (Chandler, 1923; Holy´ and Bu˚zˇek, 1965; Mai, 1985). All except one of the figured specimens of S. neglectus, S. acuticostatus, and S. headonensis plot within the area of overlap of distribution of three of the populations (polygons on Fig. 10B for HH1, BL1, and BM1). Type and figured specimens of S. websteri and S. hantonensis plot outside this area. However, type and figured specimens of all five previously named species plot within the polygon of the spread of variation for the single population from HH1 (Fig. 10B). The wide variation in the HH1 population is shown in Figure


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FIGURE 8—Representative SEM photographs of seed valves from six levels. (A) MM2, Belchato´w, Poland; reposited in the Polish Academy of Sciences. (B) BL1, Gurnard, UK; reposited in the British Museum (Natural History), London. (C) MM1, North Bohemian Basin, Czech Republic; reposited in the National Museum, Prague. (D) HM2, Bouldnor Cliff, UK; reposited in the British Museum (Natural History), London. (E) BM1, Thorness Bay, UK; reposited in the British Museum (Natural History), London. (F) HM1, Bouldnor Cliff, UK; reposited in the British Museum (Natural History), London.

10C. Furthermore, type and figured specimens of each of the three named species (S. websteri, S. hantonensis, and S. headonensis) are widely separated in Figure 10B. DISCUSSION Morphological Evolution of Stratiotes The principal-components results provide evidence of how the morphology of Stratiotes seeds has evolved

through time (Fig. 7B). The shift between levels RD1 and HH1 is due to an increase in keel width (also reflected in an increase in length/width ratio and an increase in value of the 1st eigenshape). An increase in size can be observed between HH1 and BL1 and a smaller size increase between levels BL1 and BM1. There is a large shift in morphology between levels BM1 and HM1. There is a marked decrease in overall size and a decrease in the size of the keel. This trend of decreasing size and loss of keel continues between levels HM1 and HM2. Another increase in

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FIGURE 9—Histogram stacks of three variables and the first three principal components through all eight levels. Statistical testing shows that there is no evidence of bi-modality within the data set for any variable in any level.

size occurs between HM2 and the Miocene levels (especially MM2, in which seeds exhibit the largest size of any of the levels). There also is an increase in relative keel width between HM1 and MM1 and between MM1 and MM2. The length/width ratio of the seed body changes very lit-

tle through time, which indicates that the shape of the seed body itself is not causing the changes observed in the overall shape of the seeds. These changes, therefore, must be caused by changes in the external margins of the seed coat—either by changes in the shape and relative size of the collar, keel, or the edge of the seed coat. The changes in


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TABLE 3—Type and figured specimens used in analysis; NHM No. refers to the collection numbers at the British Museum of Natural History, London; * represents holotype. In some cases, multiple specimens are catalogued under the same museum reference number, hence the multiple specimens of V.40072 and V.40079. Only one holotype is represented because in some cases the specimens are complete seeds, not valves (which are necessary for this study). Species S. S. S. S. S. S. S. S. S. S. S. S.

websteri websteri websteri websteri neglectus neglectus acuticostatus hantonensis hantonensis hantonensis headonensis headonensis

Location

NHM No.

Hamstead, IOW Hamstead, IOW Hamstead, IOW Hamstead, IOW Hamstead, IOW Hamstead, IOW Bouldnor, IOW Boscombe Bournemouth Bournemouth Hordle Hordle

V.40072 V.40072 V.40072 V.42039 V.40079 V.40079 V.40074 V.43144 V.43148 V.36484* V.42073 V.40066

size of the seed body are controlled by the overall length of the seed because the two are highly correlated. The six samples from the Hampshire Basin are relatively closely spaced geographically and are well constrained stratigraphically. They also straddle the Eocene–Oligocene boundary events (Zachos et al., 2001; Hooker et al., 2004; Grimes et al., 2005). The results obtained from the Miocene samples indicate that they are different from the samples in southern England. These samples also are not relevant for interpretation of the early evolution within the genus. For these reasons, the interpretations of evolution within the genus Stratiotes and comparisons to other biotic changes will concentrate on the six levels from southern England. Previous Evolutionary Hypotheses and their Implications Chandler (1923) studied the nine fossil species of Stratiotes that had been described at that time, and hypothesized on their possible evolutionary trends (Fig. 11). She argued the case for a single lineage from S. headonensis to S. aloides, with the exception of one offshoot in the early evolution of the genus. This subsidiary line of evolution, as she called it, included the species S. websteri, recorded from the Eocene/Oligocene transitional strata of the Hampshire Basin. This mainly anagenetic trend would indicate a gradual response to changing climatic/ecological conditions, where evolution within the genus could keep pace with the changing environment. Holy´ and Bu˚zˇek (1965) reappraised the evolution of the genus, now with 13 species. They noted two broad trends within the genus: (1) an increase in size through time; and (2) a shape change from a more globular shape in the oldest fossils to the more slender, elongate forms of more recent times. In evolutionary terms, Holy´ and Bu˚zˇek (1965) broadly agreed with Chandler (1923), but with a more branched evolutionary tree (Fig. 11). According to their interpretation, the species found in the Hampshire Basin Paleogene were on three distinct branches. The most recent portrayal of evolution within the genus Stratiotes is that of Mai (1985, fig. 13, p. 488), which showed even more branches, with rapid diversification and radiation in the Eocene followed by loss of multiple clades, resulting in only a single lineage surviving to the present day (Fig. 11). Evolution with this

Reference Chandler, Chandler, Chandler, Chandler, Chandler, Chandler, Chandler, Chandler, Chandler, Chandler, Chandler, Chandler,

1962 1962 1962 1923 1923 1923 1923 1963 1963 1963 1962 1923

Plate and Figure Plate Plate Plate Plate Plate Plate Plate Plate Plate Plate Plate Plate

35, Fig. 184 35, Fig. 184 35, Fig. 184 6, Fig. 1 5, Fig. 27 5, Fig. 27 5, Fig. 30 8, Figs. 1, 2 8, Fig. 3 7, Figs. 38, 39 27, Figs. 18, 19 5, Fig. 24

branching pattern would imply that the genus was spreading into new niches and exploiting environmental possibilities. The loss of these multiple clades through the Cenozoic, resulting in only a single species surviving to the present day, would imply that Stratiotes became restricted in some way, possibly by loss of some of the niches that it had exploited. Evolutionary Interpretations Based on This Study The results of this morphological analysis of Stratiotes have facilitated a reinterpretation of evolution within the genus, suggestive of one lineage of changing morphologies thorough time (more like that proposed by Chandler, 1923). A number of lines of evidence support this interpretation. First, each population studied exhibits a normal distribution (even though at times the intra-population variation is quite high), which indicates that at no time were there two or more coexisting species. If this pattern were to reflect different species shifting geographically with changing ecological conditions, co-occurrence of more than one species somewhere in the sequence would be expected, but this is not the case. The genus is, and always has been, endemic to Europe, which suggests that Stratiotes does not have the ability to disperse large distances in response to changing conditions. Today, there is only one species of Stratiotes, whereas other plants (e.g., Potamogeton) of similar ecology (Cook, 1990) have a number of species that can co-occur in the same area, and even in the same water body, suggesting that Stratiotes is not a taxon given to major radiations. When compared to previous studies, the results of this study concur with the broad evolutionary trends (an increase in size through time and a shape change from rounded to more elongated forms) hypothesized by Holy´ and Bu˚zˇek (1965). The Paleogene and Neogene data from this study show a reversal, with decreases in size at certain intervals, but the broad trend through time is one of increasing size. Holy´ and Bu˚zˇek (1965) described a trend of shape through time, with older species being more rounded and younger species being more elongated. The new results, however, show an initial shift in shape from elongated, with a small keel (RD1, earliest Eocene), to rounded, with a large keel (HH1, late Eocene). Following

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FIGURE 10—Type and figured specimens results. (A) Principal components plot showing position of type and figured material relative to the populations from the six sampled horizons from southern England. Polygons show the range of variation within the six sampled horizons. (B) Principal components plot showing the population range polygons of the six sampled levels as in A, sample level means with 95% confidence intervals and type and figured material of named species. (C) Scaled line drawings showing extent of variation observed within level HH1.

this initial shift there is a general trend to more elongated specimens into the Miocene. Differences in the results between this study and that of Holy´ and Bu˚zˇek (1965) are readily explained because specimens of earliest Eocene age (RD1) had not been discovered in 1965, and therefore,

the oldest specimens studied by Holy´ and Bu˚zˇek were similar in shape to the rounded specimens of HH1 (some 19 million years younger). This study included sample levels that overlap the stratigraphic ranges of the named species S. hantonensis,


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FIGURE 11—Summary of previous evolutionary hypotheses within the genus Stratiotes, redrawn from Chandler (1923), Holy´ and Bu˚zˇek (1965), and Mai (1985). Outlines of species taken from Mai (1985). Dates estimated from most-recent previous literature (Holy´ and Bu˚zˇek, 1965; Mai, 1985). Numbered species (from Chandler, 1923; Palamarev, 1979) are as follows; (1) Stratiotes headonensis, (2) S. acuticostatus, (3) S. hantonensis, (4) S. hantonensis var. bulgaris, (5) S. suborbiculatus, (6) S. neglectus, (7) S. zinndorfii, (8) S. websteri, (9) S. thalictroides, (10) S. sibiricus, (11) S. amarus, (12) S. kaltennordheimensis, (13) S. besczeulicus, (14) S. tuberculatus, (15) S. intermedius, and (16) S. aloides. See references for authorship of species not occurring in the Paleogene strata of southern England.

S. neglectus, S. headonensis, and S. websteri, which are placed on three different evolutionary branches by Mai (1985). In contrast, the data from this study indicate only one lineage in the Paleogene strata of the Hampshire Basin, evolving through time (Fig. 12). The hypothesis of Chandler (1923), with one main lineage and a single branch, is closest to the results of the current study, which has now quantified the changes along the basal part of the main lineage. Although sample levels include stratigraphic intervals from which S. websteri previously has been recorded, there is no evidence for the separate evolutionary lineage on which Chandler (1923) placed S. websteri. This study, therefore, agrees with the broad evolutionary trends proposed by Holy´ and Bu˚zˇek (1965), but not with the evolutionary hypotheses suggesting branching evolutionary histories with a number of radiations (Fig. 11) (Holy´ and Bu˚zˇek, 1965; Mai, 1985) Mean values of the samples from southern England show two clusters (Fig. 10B; HH1, BL1, BM1 and RD1, HM1, HM2), which could be taken to imply two distinct lineages. However, if two separate lineages had existed, they must either (1) have co-existed at some time somewhere (because their occurrences overlap in time), or (2) have been shifting their ranges in and out of the area. There is no evidence for two co-existing lineages in south-

ern England because all the populations are unimodal (Fig. 9, section on normality of distribution). The named species in continental Europe at this time (nos. 4 and 5 in Mai, 1985; Fig. 12 herein) are utterly different in morphology and cannot represent any of the southern English populations. Stratiotes (fossil or modern) does not occur outside Europe. Therefore, the hypothesis of two distinct lineages is rejected, with the levels studied more likely representing snapshots of a single evolutionary lineage (Fig. 12), with the HM1 and HM2 populations showing a reversal resulting in morphology similar to the RD1 population. The fact that morphologies characterizing the southern English populations are not found elsewhere in the European Eocene and early Oligocene also rules out the possibility of a migrating or shifting cline. These results indicate a single evolving lineage in the Paleogene of southern England. This lineage shows microevolution in seed morphology with anagenetic change, including one apparently more sudden morphological shift and a reversal of the evolutionary trend between samples BM1 and HM1. However, a few limitations and assumptions of the methodology and data must be mentioned. As noted above, some seed characters were omitted from this study. In each case, however, there are clear reasons why the

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FIGURE 12—Comparison of previously hypothesized evolutionary trends in Stratiotes with results from this study. Outlines of species have been taken from Mai (1985). Dates have been estimated from most-recent previous literature (Holy´ and Bu˚zˇek, 1965; Mai, 1985). Numbered species (from Chandler, 1923; Palamarev 1979) are as follows: (1) Stratiotes headonensis, (2) S. acuticostatus, (3) S. hantonensis, (4) S. hantonensis var. bulgaris, (5) S. suborbiculatus, (6) S. neglectus, (7) S. zinndorfii, (8) S. websteri, (9) S. thalictroides, (10) S. sibiricus, (11) S. amarus, (12) S. kaltennordheimensis, (13) S. besczeulicus, (14) S. tuberculatus, (15) S. intermedius, and (16) S. aloides. Circled species are those previously recorded in southern England, all in the same stratigraphic interval studied here. Drawings from this study are representative specimens of the named levels and are drawn to scale. Photographic images of example specimens can be seen in Figure 9 for comparison.

characters were unsuitable (see methods), which cast doubt on their utility as specific discriminators in the first place. The assumption is made here that seeds would change if the plant evolves. Of course, it cannot be excluded that other parts of the plant may change independently of the seeds. However, fossil leaf-margin teeth associated with the seeds are identical to those of modern Stratiotes (Collinson, 1983), suggesting stability in the vegetative structures. Furthermore, because seeds represent the whole plant at one stage of the life cycle, they are the most appropriate single-organ plant fossil to use for a microevolutionary study. The conclusions drawn here are valid only if species-specific characters are manifested in seeds. Unfortunately, owing to the extreme paucity of modern seeds, it is impossible to support or refute links between seed morphology and species. Moreover, because there is only a single modern species, its study would be of benefit only in assessing intra-specific variation. In spite of these limitations, these data nevertheless still can be used for direct comparison with the branched phylogeny models of earlier authors because these models are themselves based only on seeds.

Functional Significance of the Observed Evolutionary Changes The functional significance of the observed morphological shifts is hard to interpret. Modern Stratiotes currently set very little seed, so there is no modern analogue for comparison. Both modern and late Pleistocene seeds are very elongate and smooth (Cook and Urmi-Ko¨nig, 1983) with very narrow keels compared to Tertiary fossils (Chandler, 1923; Palamarev, 1979). The size changes observed may be related to establishment and persistence rates. An overall increase in the size of the seed also means an increase in embryo and/or storage-product volume for the next generation of the plants. A substantial body of manipulative experiments clearly shows that larger-seeded species usually perform better under hazards during seedling establishment (Westoby et al., 2002). The reason for a reduction in size of the seeds, seen in levels HM1 and HM2, is not easy to explain because the opposite for establishment and persistence would be the case. It could be caused by external factors, such as climatic effects (e.g., temperature, seasonality), which are known to


be important factors at this time in the Paleogene (Zachos et al., 2001, Lear et al., 2000) in limiting the growing season, and, hence, the ability of the plant to produce large seeds. Another possible reproductive strategy would involve the production of a greater number of smaller seeds (equivalent to fewer larger seeds per unit effort). An increased number of smaller seeds gives an increased chance of encountering more diverse growth sites and could reflect increasing habitat variation and/or a disturbance in the local environment. This reversal to a smallseeded strategy occurs in the lower Hamstead Member immediately prior to the Oi-1 glaciation (Hooker et al., 2004), and coincides with a reversal to narrower seed keels. The change in the relative size of the keel of the seeds is an important factor in the overall seed shape changes, but is again hard to explain in terms of functional significance. There is an obvious potential hydrodynamic role for the keel. Changes in the size of the keel could cause subtle changes in how the seeds float, orient, and move in the water, which could affect dispersal. The germination dehiscence zone passes through the keel, and keel size might influence seed orientation on the sediment-water interface. Thus, keel variation might influence germination behavior. There also is a possibility that changes in the keel are caused by the arrangement, number, or packaging of the seeds within the fruit, but lack of modern comparative material makes this hard to assess. It is important to note that the broad trend of elongation of the seeds through time is effectively a reduction in the size of the keel, so any adaptive value of larger keels was restricted to a short interval (HH1, BL1, and BM1). Relationship to Other Biotic Turnover The results can be compared with those of a similar study (Sille et al., 2004) on charophytes (aquatic green algae). A marked shift in shape and size (representing a doubling in the volume of the gyrogonite) of the charophyte genus Harrisichara can be observed between the Headon Hill Formation and the Bembridge Limestone Formation (Fig. 13; Sille et al. 2004). The size increase observed in Stratiotes over this time interval is not as marked as with the charophytes. In the latest Eocene and earliest Oligocene, the morphological trends can be seen to decouple. The morphology and size of the charophytes remain stable through this time period, whereas Stratiotes reduces in size and the relative size of the keel decreases. Sille et al. (2004) argued that morphological evolution in Harrisichara was the result of the climatic fluctuations observed in the late Eocene and early Oligocene. Therefore, the decoupling of the morphological trends could be the result of either slightly different ecologies of the plants, or a different way of evolving to cope with climate fluctuations. Thus, the charophytes underwent one distinct and relatively rapid change, whereas Stratiotes changed gradually through the latest Eocene and earliest Oligocene. There are a number of mammal faunal turnover events in the late Paleogene strata of the Hampshire Basin. The largest is the ‘Grande Coupure’ in the earliest Oligocene (Hooker et al. 2004); another is in the Bembridge Limestone Formation (Hooker et al. 1995). There also is a small

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rodent turnover event slightly pre-dating the ‘Grande Coupure’ (Hooker et al., 2004). A possible factor linking mammal faunal turnover and evolution within Stratiotes recently has been observed (Collinson and Hooker, 2000), where a number of Stratiotes seeds have been found with rodent gnaw marks in them. These seeds were found in the Headon Hill Formation and Bembridge Limestone Formation, but not higher in the stratigraphy. No evidence of predation has been found during this study on the smaller seeds with narrow keels in the Hamstead Member. Predation of larger seeds could result in selection favoring smaller seeds so that a decrease in overall size of the seed could be a response to rodent predation. A smaller keel also might render the seed harder for the rodent to manipulate or orientate. Discussion of Type and Figured Material Type and figured specimens of previously named Paleogene species from southern England are not closely juxtaposed to the population means (Fig. 11B). The seed populations cannot be assigned to species within the current taxonomic framework. Figured specimens of all five named Paleogene species plot within a polygon (Fig. 11B) representing the spread of variation of a single population from this study (HH1). In addition, specimens of at least two named species lie within the range polygon of each sample level. This suggests that a taxonomic reappraisal may be necessary, with the currently named species merely representing components of the variation of a population. The taxonomic splitting of currently named species may have influenced some authors to propose evolutionary hypotheses of branching clades, in contrast to the single lineage recognized in this study. However, the methodology used in this study cannot be applied to complete seeds, so it was impossible to include the holotypes of all of the named species, and thus a formal revision of the taxonomy would be premature. Meanwhile, the single lineage recognized here can be referred to informally as the Stratiotes headonensis lineage using the name of the basal and first-described species in the main lineage first proposed by Chandler (1923). CONCLUSIONS (1) The morphometric methodology used in this study has demonstrated quantifiable microevolution within the genus Stratiotes. (2) This research indicates that one evolving lineage is more likely as an evolutionary hypothesis for the early history of the genus in the Paleogene than previous multiple-lineage hypotheses. (3) The changes observed in seed shape are dominantly controlled by alterations in relative keel size. (4) The functional significance of the observed morphological changes is very difficult to explain satisfactorily, and the lineage shows reversal to smaller size and narrower keels. Hydrodynamic effects of differing keel sizes and differing reproductive strategies (involving differing size of seeds and storage products) could have played a role. (5) The decoupling of morphological evolution between Stratiotes and the charophyte genus Harrisichara

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FIGURE 13—Comparison of Stratiotes evolution with that of the charophyte genus Harrisichara (Sille et al., 2004), and with important global and local events across the Eocene–Oligocene transition (Collinson and Hooker, 2000; Hooker et al., 2004).

across the Eocene–Oligocene transition may indicate a role for animal/plant interactions in evolution within the genus Stratiotes. (6) Assessment of type and figured material of named species against population means obtained in this study indicates that a taxonomic reappraisal may be necessary. Taxonomic splitting may have led to previ-

ous evolutionary hypotheses of branching clades that are not supported by this study. ACKNOWLEDGEMENTS The work for this paper was carried out by NPS during a Ph.D. studentship funded by the Isle of Man Govern-


ment and the Natural History Museum (CASE Partner). We would like to thank Andy Currant (NMH London), Steve Grimes (Plymouth), and Dave Mattey for assistance with fieldwork; David Bone for providing specimens from Felpham; Sharon Gibbons for assistance with lab work; Kevin D’Souza and Andy Henderson (NHM London) for assistance with photography; and Jiri Kvacˇek (NM Prague), Ewa Zastawniak (Polish Acad. Sci.), Paul Kenrick, and Cedric Shute (NHM London) for specimen loans. NPS would like to thank Dani Schmidt and John Wright for helpful discussions. MEC would like to thank Robert Scotland and Tony Brain for their major contributions to a pilot study many years ago, which enabled her to appreciate the potential for the project that forms the basis of this paper. We would also like to thank Kathleen Pigg for her helpful review of the manuscript and a second, anonymous reviewer. REFERENCES BERGGREN, W.A., KENT, D.V., SWISHER, C.C., III, and AUBRY, M.-P., 1995, A revised Cenozoic geochronology and chronostratigraphy: in Berggren, W.A., Kent, D.V., Aubry, M.-P., and Hardenbol, J., eds., Geochronology, Time Scales and Global Stratigraphic Correlation: Society of Economic Paleontologists and Mineralogists Special Publication 54, p. 129–212. BONE, D.A., 1986, The stratigraphy of the Reading Beds (Palaeocene), at Felpham, West Sussex: Tertiary Research, v. 8, p. 17–32. CHANDLER, M.E.J., 1923, Geological history of the genus Stratiotes: Quarterly Journal of the Geological Society of London, v. 79, p. 117–138. CHANDLER, M.E.J., 1957, The Oligocene flora of the Bovey Tracey Lake Basin, Devonshire: Bulletin of the British Museum of Natural History, Geology, v. 3, p. 71–123. CHANDLER, M.E.J., 1960, Plant Remains of the Hengistbury and Barton Beds: Bulletin of the British Museum of Natural History, Geology, v. 4, p. 191–238. CHANDLER, M.E.J., 1961, Flora of the Lower Headon Beds of Hampshire and the Isle of Wight: Bulletin of the British Museum of Natural History, Geology, v. 5, p. 93–157. CHANDLER, M.E.J., 1962, The Lower Tertiary floras of Southern England: Vol. II, Flora of the Pipe Clay series of Dorset (Lower Bagshot): British Museum of Natural History, London, 176 p. CHANDLER, M.E.J., 1963, Revision of the Oligocene floras of the Isle of Wight: Bulletin of the British Museum of Natural History, Geology, v. 6, p. 323–383. COLLINSON, M.E., 1983, Palaeofloristic assemblages and Palaeoecology of the Lower Oligocene Bembridge Marls, Hamstead Ledge, Isle of Wight: Botanical Journal of the Linneaen Society, v. 86, p. 177–225. COLLINSON, M.E., 1990, Plant evolution and ecology during the early Cainozoic diversification: Advances in Botanical Research, v. 17, p. 1–98. COLLINSON, M.E., and CLEAL, C.J., 2001, The Palaeobotany of the Palaeocene–Eocene transitional strata in Great Britain: in Cleal, C.J., Thomas, B.A., Batten, D.J., and Collinson, M.E., eds., Mesozioc and Tertiary Palaeobotany of Great Britain: Geological Conservation Review series, 22, p. 155–184. COLLINSON, M.E., and HOOKER, J.J., 2000, Gnaw marks on Eocene seeds: evidence for early rodent behaviour: Palaeogeography, Palaeoclimatology, Palaeoecology, v. 157, p. 127–149. COLLINSON, M.E., HOOKER, J.J., and GROCKE, D.R., 2003, Cobham lignite bed and penecontemporaneous macrofloras of southern England: a record of vegetation and fire across the Paleocene–Eocene thermal maximum: in Wing, S.L., Gingerich, P.D., Schnitz, B., and Thomas, E., eds., Causes and Consequences of Globally Warm Climates in the Early Paleogene: Geological Society of America Special Paper 369, p. 333–350. COLLINSON, M.E., SINGER, R.L., HOOKER, J.J., 1993, Vegetational change in the latest Eocene of southern England: in Planderova,

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