Heteropolar eunotioid diatoms (Bacillariophyceae) were common in the North American Arctic during the middle Eocene

Eunotioid diatoms that express asymmetry in both the apical and transapical axes, forming heteropolar valves, are generally placed in the genus Actinella. The degree of heteropolarity varies between species, ranging from subtle differences between poles to highly differentiated head poles bearing an apical protuberance. Actinella species with less difference between the poles and lacking an apical protuberance are gradational with Eunotia. With over 100 known species reported globally, primarily in the tropics, only Actinella punctata Lewis, 1864 is currently known from North America. As part of a biotic survey and inventory project focused on the middle Eocene Giraffe crater locality near the Arctic Circle in northern Canada, we have uncovered a wealth of eunotioid diatoms including at least five heteropolar species attributed to Actinella, three of which are described formally herein as A. hickeyi sp. nov., A. goodwinii sp. nov. and A. kimberlitica sp. nov.. These diatoms all lack apical protuberances and bear resemblance to modern heteropolar counterparts within Eunotia. The objectives of this contribution are to report the findings from the Giraffe locality relative to modern and fossil eunotiophycid taxa, discuss the use of heteropolarity as a distinguishing character for the genus Actinella, and consider the palaeoclimatic and biogeographical implications of these observations.

Eunotia papilioforma Furey, Lowe & Johansen, each bearing slightly club-shaped and heteropolar valves. Sabbe et al. (2001) noted that valves of E. rhomboidea could be isopolar or heteropolar and noted that placement in Eunotia requires further study. Furey (2011) clearly stated that valve shape and position of the helictoglossae in E. papilioforma are asymmetrical to the transapical axis, but still placed the taxon in Eunotia despite also noting that Actinella species are distinguished from Eunotia by heteropolar valve shape. Placement of E. papiloforma under Eunotia, rather than Actinella, given their distinguishing criteria, is unclear and this taxon may more appropriately belong in Actinella. Regardless of the generic assignment of E. rhomboidea and E. papiloforma, it is clear based on the hundreds of diatom studies performed throughout North America over the last century that heteropolar Eunotiophycidae species are extremely rare. A similar case has been made globally (see Sabbe et al., 2001).
The Giraffe fossil locality (64°48′N, 110°04′W) is a kimberlite diatreme crater that formed following phreatomagmatic volcanism, subsequently infilled with a sequence of Eocene lacustrine and then paludal sediments, and was later entombed with Neogene glacial deposits (Wolfe et al., 2006;. A 165 m long drilled core was retrieved from the crater by BHP Billiton inc. during diamond exploration and later archived at the Canadian Geological Survey (Calgary). The core contains over 40 m of terrestrial sediment atop 65 m of lacustrine mudstone of middle Eocene age (Wolfe et al., 2006;). Both the terrestrial (doria et al., 2011) and lacustrine (Siver & Wolfe, 2005;Wolfe et al., 2006) deposits contain abundant fossil remains with extraordinary preservation, including numerous freshwater diatoms among other siliceous microfossils Siver et al., 2010). As part of a biotic survey and inventory project focused on the lacustrine mudstones we have uncovered a wealth of Eunotiophycidae diatoms, mostly contained within a 16 m section of the core, and representing species of Eunotia and Actinella. of special interest is the presence in this single fossil locality of five or more species each exhibiting a heteropolar condition and that are most appropriately classified as Actinella. The purpose of this contribution is to report our findings to date and formally describe three of the species.

MAtErIAls And MEtHods
Mudstone chips (50-100 mg) from eleven sections of the Giraffe core spanning 8.81 m (Table 1) were oxidized using 30% H 2 o 2 under low heat for a minimum of an hour, rinsed with distilled water, and the resulting slurries stored in glass vials. This procedure results in separation of many siliceous microfossils from the mudstone matrix. in addition, small fragments of mudstone, each containing many embedded microfossils, remain within each preparation. Aliquots of each slurry were air dried on to pieces of heavy duty aluminum foil and directly on to circular glass coverslips. The aluminum foil samples were trimmed, attached to aluminum stubs with Apiezon® wax, and coated with a mixture of gold and palladium for 2 min with a Polaron Model E sputter coater. Samples were examined with a Leo 982 field emission or an FEi Nova scanning electron microscope (SEM). The coverslips were mounted on to glass slides with Naphrax and examined with a Leica dMR or an olympus Bx51 light microscope (LM). A minimum of 20 specimens for each species were used for morphometric analysis.
Each sample from the Giraffe core is identified with a threepart number (Table 1). The first number represents the core box. deeper sections of the core correspond to larger box numbers. Each box contains three 1.5 m core lengths, identified as channels 1, 2 and 3. The second number represents the channel. The third number indicates the length in centimetres measured down from the top of a core length. For example, sample 14-3-100 represents a sample taken from 100 cm down along the core length in channel 3 from box 14.

rEsults
Eunotiophycidae specimens from the Giraffe locality represent at least five, and probably six, different species with heteropolar valves (Pls 1-6). one of these species, Actinella giraffensis, has been previously described (Siver et al., 2010), and we now have examined a sufficient range of specimens to describe three additional species formally. Remains of two more species have also been uncovered, but since we have examined a limited number of specimens to date, only preliminary observations are given herein.   remarks. Actinella hickeyi sp. nov. was found in four sections, spanning over 8 m of the core (Table 1) Pl. 5, figs A-E). one apex, the foot pole, is narrower, slightly subcapitate and extended further than the other apex, or head pole, yielding a heteropolar condition. The undulations, especially the one towards the foot pole, are less pronounced on smaller valves. Valves range in size from 16-36 µm × 4.4-6.5 µm. The mantle is wide and forms a right angle with the valve face (Pl. 5, figs A-d) except around the foot pole where it is slightly wider, angled outward and extended past the valve face (Pl. 5, fig. A, arrow). This difference in the width and angle of the mantle between each pole often yields a slight rhomboidal shape in girdle view, more pronounced on the foot pole end of the frustule (Pl. 5, fig. d).
The striae are evenly spaced, parallel over most of the valve, become slightly radiate around the apices, and range from 16 to 20 in 10 µm (Pls 2 and 5). The striae on the valve face align roughly with those on the mantle; however, there are often additional shorter striae on the mantle (Pl. 5, figs A and d). There is a series of short and more closely spaced striae between the raphe slits and the lower edge of the mantle (Pl. 5, figs C-d). The areolae are very small, circular, evenly spaced pores that lack coverings on both the external and internal surfaces (Pl. 5). The raphe slits are short, terminated on each end with small pores, and are positioned on the mantle (Pl. 5, figs B-d). The proximal end of the raphe is situated about one-third of the distance from the valve face, traverses up to the valve margin, and terminates a short distance on to the valve face (Pl. 5, figs B-d). internally, the distal ends of the raphe slits terminate as small helictoglossae along the ventral margin (Pl. 5, figs E-F). The helicoglossa on the foot pole terminates further from the apex than the one on the head pole. There is one rimoportula per valve positioned on the foot pole apex slightly towards the dorsal margin (Pl. 5, figs E-F, arrows). The lower margin of the mantle is thickened around each apex, especially around the foot pole where it is extended inward forming a shallow pseudoseptum (Pl. 5, fig. F). There are three to four wide, open and wavy girdle bands per frustule, each with well-developed striae.
remarks. Actinella goodwinii was found in six closely spaced sections spanning 1.6 m of the core (Table 1). These sections were largely dominated with scaled chrysophyte remains, including especially high numbers of a species resembling Mallomonas lychenensis, M. porifera and chrysophyte cysts, other eunotioid diatoms, the diatom Oxyneis apporrecta Siver, Wolfe & Edlund, and euglyphids.  6, figs A-C). The dorsal margin is slightly convex, the ventral margin is straight and the apices are rounded. The foot pole is narrower and extends further relative to the helictoglossa than the head pole (Pl. 1, figs i-o; Pl. 6, figs A-B, arrows). There is a narrow, but distinct, axial area aligned along the ventral valve margin that connects the distal raphe ends (Pl. 6, fig. A). The striae are closely and evenly spaced across the entire valve, parallel with the transapical axis, and range in density from 22-26 per 10 µm. The areolae are small, circular, evenly spaced pores that lack coverings (Pl. 6; figs A-C).
The raphe slits are situated on the mantle and gradually bend up on to and terminate on the valve face (Pl. 6, figs A-B). Externally, the distal and proximal raphe fissures terminate as small pores. internally, the distal end of the raphe terminates as a well-formed helictoglossa bent slightly towards the dorsal margin, yielding an incision-like appearance when viewed with a light microscope (Pl. 1, figs i-o). There is one rimoportula per valve apically positioned on the foot pole (Pl. 6, fig. C,  arrow).
remarks. Actinella kimberlitica was found in five sections spanning almost 7 m of the core (Table 1). These sections were largely dominated with scaled chrysophyte remains, including a species resembling Mallomonas lychenensis and M. porifera, chrysophyte cysts, numerous other eunotioid diatoms, the diatom Oxyneis apporrecta, and euglyphids. Additional Actinella taxa Specimens of two additional heteropolar forms representing Actinella were recovered from the Giraffe locality, including a long slender diatom designated provisionally as Actinella sp. 1 (Pl. 6, figs d-F), and a small club-shaped form designated as Actinella sp. 2 (Pl. 1, figs P-Q). We note their presence in the core, but elect not to officially describe them at this time until additional specimens can be investigated to establish their full range of characters. However, the presence of these additional taxa provides an expanded perspective of the full diversity of eunotiophycid diatoms from the deposit.
Actinella sp. 1. Specimens of this taxon ranged between 50 and 55 µm in length, and have heteropolar valves that taper gradually from about 5 µm wide at the head pole to 3 µm wide at the foot pole (Pl. 6, fig. d). A slight bend in valve shape occurs along the dorsal margin closer to the foot pole, and both apices are rounded. The mantle forms a right angle with the valve face except around the foot pole where it is angled outward and extended past the valve face (compare Pl. 6, figs E and F). There is a distinct axial area aligned along the ventral valve margin that connects the distal raphe ends (Pl. 6, figs d and F, white arrows). Striae are closely and evenly spaced, parallel with the transapical axis and composed of tiny areolae that lack occlusions at least on the external surface. Striae are continuous on to the mantle, and become radially aligned around the apices (Pl. 6, figs E and F). The raphe is straight, runs diagonally along the mantle to the valve margin, and bends sharply up onto and terminates midway on the valve face. The raphe on the head pole is more sharply angled onto the valve face and the distal fissure terminates closer to the apex than on the foot pole (compare Pl. 6, figs E and F). Each distal fissure is surrounded by a well-developed hyaline region. Although we have not yet been able to examine internal structure, we believe this taxon has at least one rimoportula situated on the foot pole (Pl. 6, fig. F, black arrow). Based on limited observations, frustules are slightly clavate in girdle view (not shown). The overall valve morphology and the structure of the raphe appear to distinguish this taxon from all other described Actinella species.
Actinella sp. 2. Valves are small, range in length from 10-14 µm to 2.5-3.5 µm, and are distinctly club-shaped with a wide head pole and narrow foot pole (Pl. 1, figs P-Q). Apices are rounded, and the striae are closely spaced and parallel with the transapical axis. it is possible that these specimens represent very small valves of Actinella giraffensis (Siver et al., 2010); however, since we have not found similar-shaped valves of this size in material containing numerous specimens of the later taxon, it likely represents a new species.

dIscussIon
The position and reduced nature of the raphe, presence of rimoportulae, and features of the striae, areolae and helicoglossae are similar for Actinella and Eunotia, and both genera are, without doubt, evolutionarily closely related (Round et al., 1990;Kociolek, 2000). The primary, and to date perhaps only valid, character separating Actinella from Eunotia is the heteropolar shape of the valve (Round et al., 1990;Sabbe et al., 2001;Melo et al., 2010;Ripple & Kociolek, 2013). By definition, valves of Eunotia are symmetrical to the transapical axis (Eunotia rhomboidea and a few related taxa being the exceptions, as stated previously), while those of Actinella are not, yielding the heteropolar condition. As noted by Sabbe et al. (2001), heteropolarity is the obligatory character for inclusion in Actinella, and this is the primary criterion on which we ascribe the new species from the Giraffe pipe as belonging to Actinella. in our opinion, given the current definitions of both genera, the new species are best classified as Actinella.
Although all Actinella species are heteropolar in valve view, polarity in girdle view remains an open question. Many Actinella species are also heteropolar in girdle view forming clavate or wedge-shaped frustules (Sabbe et al., 2001;Siver et al., 2010). However, a few species have been noted to be rectangular or linear in girdle view, and sometimes narrower in the middle of the frustule (Lewis, 1864;Patrick & Reimer, 1966;Metzeltin & Lange-Bertalot, 1998). Also, as noted by Sabbe et al. (2001), polarity in girdle view is not always given in original descriptions and this character needs to be determined for many Actinella species before a thorough study can be made for the genus. Although we believe frustules of Actinella hickeyi are indeed slightly clavate in girdle view based on observations of a few specimens with light microscopy, whole frustules have proven difficult to find and we have not been able to examine this character thoroughly with SEM despite observing hundreds of isolated valves.
Based on our current observations, Actinella goodwinii is also heteropolar in girdle view (i.e. clavate), with a somewhat wider and rectangular-shaped head pole, and a slightly rhomboidalshaped and narrower foot pole. This morphology is consistent with the different shapes found at each valve pole, and the rhomboidal-shape of the foot pole is reminiscent of that observed on some Eunotia taxa, particularly Eunotia rhomboidea Hustedt. Valves of Eunotia rhomboidea can be isopolar or heteropolar in valve view and, as noted by Sabbe et al. (2001, p. 337), 'heteropolarity in Eunotia cells is the exception rather than the rule'. Since valves of A. goodwinii are always heteropolar in valve view, this taxon is best described as Actinella.
The degree of heteropolarity found in Actinella species ranges from very slight to extreme. Many species of Actinella, such as A. punctata Lewis and A. guianensis Grunow, produce valves with uniquely shaped and highly inflated head poles relative to the foot pole. This difference is taken to extremes in taxa such as A. muylaertii Sabbe & Vyverman and A. comperei Sabbe, Vanhoutte & Vyverman, where the foot pole is more or less reduced to a narrow point with a width of less than 2 µm (Sabbe et al., 2000;. However, there are a fair number of Actinella species that bear only a slight heteropolarity and at first glance could be mistaken for Eunotia. For example, Actinella indistincta Vyverman & Bergey, A. pararobusta Metzeltin & Lange-Bertalot, A. parva Vanhoutte & Sabbe and A. pareunotioides Metzeltin & Lange-Bertalot display limited heteropolarity in valve view, possess valves that lack inflated poles, and are 'Eunotia-like'. in fact, some species were initially described as Eunotia, only later being transferred to Actinella based on the heteropolar nature of the valve (Metzeltin & Lange-Bertalot, 2007). other species, such as A. gessneri Hustedt and A. siolii Hustedt, have very inflated head and foot poles and display only slight heteropolarity in the transapical axis. Still, other species were initially described belonging to Asterionella Hassall, only later discovered to possess reduced raphes, and transferred to Actinella (Kociolek & Rhode, 1998  rionegrensis Metzeltin & Lange-Bertalot as being commonly isopolar with a rare number of valves displaying a slight heteropolarity. These authors further expressed difficulty in assigning this taxon to a given genus. Since heteropolarity is rare and not the norm for A. rionegrensis, it may best be placed in Eunotia or Desmogonium (see Sabbe et al., 2001). We view the degree of heteropolarity expressed in A. hickeyi and A. goodwinii to be slight, but clearly distinct, present on all valves, and within the range expressed by numerous species currently included in Actinella.
Should the heteropolar character be used to distinguish between the genera Actinella and Eunotia? This question, reviewed by Sabbe et al. (2001), has a rather long history, with some authors (e.g. Cholnoky, 1954;Metzeltin & Lange-Bertalot, 1998) arguing for placement of Actinella at the subgeneric rank under Eunotia, and thus demoting the use of heteropolarity as a distinguishing character at the generic level. in fact, Metzeltin & Lange-Bertalot (1998) described a distinctly heteropolar taxon as Eunotia falcifera Metzeltin & Lange-Bertalot, and further placed it in the proposed subgenus Cultria under the genus Eunotia. Cultria was to accommodate heteropolar species with head poles that are more or less rounded and lack an apical protuberance. However, Metzeltin & Lange-Bertalot (2007) later retracted this position and transferred Cultria, along with E. falcifera, to Actinella. As already noted, many Actinella species are morphologically closely allied with Eunotia counterparts, such as A. aotearoaia Lowe, Biggs & Francoeur, A. parva, A. indistincta, A. eunotioides Hustedt, A. robusta Hustedt, A. peronioides Hustedt, and the Giraffe taxa described in this paper and earlier by Siver et al. (2010).
Heteropolarity is likely an adaption for attachment, either directly on to a substrate, through formation of a mucilaginous stalk (e.g. Actinella aotearoaia), or to each other to form a colony (Sabbe et al., 2001). in fact, in his original description of Actinella, Lewis (1864) recorded stellate colonies of A. punctata, with cells connected to each other by their foot poles. one of us (PAS) has observed such colonies of A. punctata in plankton samples from Lewis' type locality, Saco Pond. if a heteropolar shape facilitates attachment, and if attachment would prove beneficial for survival, then one possible scenario is that the heteropolar condition evolved multiple times within Eunotia, yielding a suite of species now classified as Actinella. Such a hypothesis would explain why some Actinella taxa have morphologically similar counterparts in Eunotia. As discussed below, we certainly recognize this possibility with respect to the relationship between Actinella goodwinii and Eunotia bidentula W. Smith. if this scenario is proven correct, Actinella would represent a polyphyletic genus in need of re-evaluation.
Combining Actinella giraffensis (Siver et al., 2010) with the results of this study, there are now at least five, and probably six, Actinella species known from the Giraffe locality. This finding is especially intriguing from three points of view. First, there are more Actinella species in this single Eocene locality than known today from all of North America and Europe Ripple & Kociolek, 2013). in addition, a review of the literature for Eunotia shows that heteropolar specimens are extremely rare (Sabbe et al., 2001), making the range of heteropolar eunotiophycid taxa in the Giraffe material even more impressive and unique. Second, the high-latitude geographical location of the Giraffe locality is far removed from the modern centres of biodiversity for Actinella in the Southern Hemisphere (Kociolek et al., 2001;Sabbe et al., 2001), which is especially pronounced in low pH brown-water (humic) ecosystems of the tropics (Melo et al., 2010). These observations support the hypothesis proposed by  that this genus was historically much more widespread than at present in the Northern Hemisphere. This hypothesis is further supported by the fact that the only other known fossil finds for Actinella are from the Northern Hemisphere, having being uncovered from eastern Russia (Actinella penzhica; Lupikina & dolmatova, 1984), France (Actinella pliocenica; Héribaud, 1902), and the northern Province of Jiling in China (Actinella miocenica; Li, 1988). Third, it is equally intriguing that these Eocene taxa lack highly heteropolar Actinella species and ones with apical protuberances, as well as more elaborately shaped Eunotia taxa similar to ones found today especially in South America (Metzeltin & Lange-Bertalot, 1998;. it is tempting to suggest that the Eocene may represent a time period where Actinella was beginning to diverge from Eunotia by evolving a heteropolar condition more adapted for an attached life style, and prior to the time where both genera began to develop more elaborate valve morphologies. Future molecular phylogenetic studies will undoubtedly help elucidate the evolutionary histories of these two closely related genera and determine their generic validity. The morphological structure of Actinella goodwinii is similar to Eunotia bidentula (Siver & Hamilton, 2011), and it is possible that the two taxa shared a common ancestor. The overall valve shape, structure of the raphe, striae pattern and wide girdle bands are very similar in both species (Siver & Hamilton, 2011). in addition, Siver & Hamilton (2011) illustrated a similar apical structure for E. bidentula as found on A. goodwinii, and the base of the mantle around the apices is thickened on both taxa. However, the consistent heteropolar nature of Actinella goodwinii specimens clearly distinguish it from the modern E. bidentula. in addition, valves of A. goodwinii have a slight, but clearly discernable, concave ventral surface and frustules with foot poles that are slightly rhomboid in girdle view. The ventral surface on E. bidentula specimens is straight and the frustules rectangular in girdle view. Eunotia bidentula specimens are also larger, have more pronounced capitate apices, and have a longer raphe. Lastly, the distal raphe fissure terminates at the valve margin on E. bidentula (Siver & Hamilton, 2011), whereas on A. goodwinii specimens it bends up and extends on to the valve face. in our opinion, the slight modifications on A. goodwinii that yield its heteropolar nature and reduced raphe structure may be the result of adaption to an attached life style.
Actinella goodwinii also bears some resemblance to Eunotia camelus Ehrenberg, E. cameliopsis Metzeltin & Lange-Bertalot, E. schneideri Metzeltin & Lange-Bertalot and E. diodon Ehrenberg, but can be distinguished from each based on the heteropolar nature of the frustules, differences in valve morphology and raphe structure. Specimens of E. camelus and E. cameliopsis have concave ventral margins and two undulations along the dorsal margin similar to A. goodwinii. However, on larger specimens of both E. camelus and E. cameliopsis the number of dorsal undulations increases and becomes less distinct. in addition, the distal raphe end of E. camelus encircles the apex, and specimens of E. cameliopsis possess widely spaced striae. Relative to Maps are easy to integrate into GIS, presentation software, and other analysis tools The Geofacets-GSL

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