Revision of the non-marine centric diatom flora (Bacillariophyta) of the sub-Antarctic Campbell Island (southern Pacific Ocean) with the descriptions of five new species

During a survey of the moss-inhabiting diatom flora of the sub-Antarctic Campbell Island, located in the southern Pacific Ocean, several unknown centric diatoms were observed that could not be identified using the currently available literature. Detailed light and scanning electron microscopical observations and comparisons with the characters of several species of Melosira, Angusticopula, Ferocia and Arcanodiscus worldwide indicated that five of them should be described as new to science: Angusticopula cosmica Goeyers & Van de Vijver sp. nov., Arcanodiscus crawfordianus Goeyers & Van de Vijver sp. nov., A. indistinctus Goeyers & Van de Vijver sp. nov., A. saundersianus Goeyers & Van de Vijver sp. nov. and Ferocia houkiana Goeyers & Van de Vijver sp. nov. A sixth species, Angusticopula chilensis, was illustrated for the first time using SEM and as a result is considered to differ sufficiently from A. dickiei to warrant epitypification as A. chilensis. The new species were first described in genera that formerly were included within the genus Melosira. All six species are morphologically characterized and compared with similar species within their respective genera. Their presence and distribution on Campbell Island are discussed based on the observations made in the available samples.


Introduction
The non-marine diatom flora of the sub-Antarctic Islands in the southern Pacific Ocean has received far less attention than the flora in the (sub-)Antarctic parts of the Indian and Atlantic Oceans (see for instance Van de Vijver et al. 2002;Zidarova et al. 2016 and references therein). The Pacific sector of the sub-Antarctic region comprises only a few small islands: Macquarie Island (128 km 2 ), Snares Island (3.3 km 2 ), Auckland Island (626 km 2 ), Campbell Island (113 km 2 ), Antipodes Island (21 km 2 ) and Bounty Island (1.35 km 2 ) (Chown et al. 1998). Only a few studies have been published on the diatom flora of these islands (Van de Vijver & Beyens 1999). Most published information comes from The diatom flora of Campbell Island was studied by Hickmann & Vitt (1973) who found 59 taxa living epiphytically on mosses. A second study in 2015 by Saunders et al. analyzed the relationship between aquatic diatom communities and water chemistry in 54 lakes and ponds on the island. They observed a high floristic similarity between Campbell Island and the sub-Antarctic Islands in the southern Indian Ocean ( Van de Vijver et al. 2002) and with the flora in the Tasmanian and New Zealand mountain lakes (Vyverman et al. 1995;Hodgson et al. 1997;Kilroy 2007;John 2018).
During a visit to the moss herbarium at the British Antarctic Survey (Cambridge, UK), the original moss samples that were collected by Dale Vitt and used in their diatom survey (Hickmann & Vitt 1973) were retrieved and subsampled in the BAS herbarium. After preparation for diatom analysis, it was clear that the diatom flora in these moss samples was very well preserved. Analysis of the slides revealed the presence of several centric taxa that could not be identified using the currently available literature.
The genus Melosira was originally published in 1827 by Agardh but it was Kützing (1844) who included a large number of taxa in the genus. Later authors revised parts of the genus and split off several, often newly described, genera such as Aulacoseira Thwaites (1848), Orthoseira Thwaites (1848), Paralia Heib. (Heiberg 1863) and Ellerbeckia R.M. Crawford (1988). Nevertheless, a large number of taxa remained in the genus Melosira. In 2017, the genus Melosira was revised, which resulted in the description of several new genera: Ferocia Van de Vijver & Houk (in Van de Vijver et al. 2017), Angusticopula Houk et al. (2017) and Arcanodiscus Maidana & E.Morales (in Maidana et al. 2017). The description of the new genera resulted in a revision of all diatom taxa belonging or formerly belonging to the genus Melosira in the sub-Antarctic region. Four new taxa were described: Ferocia ninae Van de Vijver (in Van de Vijver et al. 2017), F. subantarctica Van de Vijver & Houk (2019), Arcanodiscus desmetianus Van de Vijver (in Van de Vijver & Houk 2019) and Melosira jeanbertrandiana Van de Vijver & Crawford (2019) whereas the correct taxonomic identity of two other taxa was established: Angusticopula dickiei (Thwaites) Houk et al. and A. chilensis (Grunow) Houk et al. . These revisions, however, only concerned those taxa that were observed on the islands in the southern Indian Ocean. The recent survey of the Campbell Island moss samples partly fills this gap in our knowledge of the sub-Antarctic melosiroid diatom flora.
Five unknown taxa were found and after a thorough analysis of light and scanning electron microscopy observations and comparison with previously described taxa worldwide, they are described as new to science: Angusticopula cosmica sp. nov., Ferocia houkiana sp. nov., Arcanodiscus crawfordianus sp. nov., A. indistinctus sp. nov. and A. saundersianus sp. nov. Several populations of a sixth species, Angusticopula chilensis, were analyzed in more detail and compared with published images of the Grunow type slide (in Houk et al. 2017: pl. 63, figs 1-6). As unmounted material of this taxon is no longer available (A. Igersheim, Vienna, pers. comm.), one of the Campbell Island populations will be designated as epitype for this species. In the present paper, the morphology of the new species is discussed and the species are compared with similar species worldwide and more specifically with taxa described from the sub-Antarctic region.

Material and methods
Campbell Island (52°32′24″ S, 169°8′42″ E) is the main island of a small volcanic archipelago situated in the southern Pacific Ocean, at approx. 600 km south of New Zealand and 3800 km north of the Antarctic Continent. The island has a total surface area of 113 km 2 , reaching an altitude of 569 m. Saunders et al. (2015) provide details on the climate, geomorphology and vegetation of the island.
During a National Science Foundation expedition between December 1969 and February 1970, a large number of moss samples were collected by Prof. Dale Vitt (Southern Illinois University Carbondale, USA) (Hickmann & Vitt 1973;Vitt 1974 Subsamples of the selected material were prepared for light microscopy (LM) observation following the method described in Van der Werff (1955). Small parts of the samples were cleaned by adding 37% H 2 O 2 and heating to 80°C for about one hour. The reaction was completed with the addition of saturated kmnO 4 . After digestion and centrifugation (3 × 10 minutes at 3700 g), the material was diluted with distilled water to avoid excessive concentrations of diatom valves. Cleaned diatom valves were mounted in Naphrax®. Samples and slides are stored at the BR-collection (Belgium). The slides were analyzed using an Olympus BX53 microscope, equipped with Differential Interference Contrast (Nomarski) and the UC30 camera connected to the Cell Sense Standard program. For scanning electron microscopy (SEM), part of the suspension was filtered through polycarbonate membrane filters with a pore diameter of 1 μm, pieces of which were fixed on aluminum stubs after air-drying. The stubs were sputter-coated with 10 nm of Pt and studied in a JEOL JSM-7100F at 2 kV (Meise Botanic Garden, Belgium). For each new taxon, the number of specimens measured at random on the type slide is indicated (n = X). Terminology follows Round et al. (1990), Maidana et al. (2017), Van de Vijver et al. (2017) and Houk et al. (2017). The morphology of the new species was compared with species described or discussed in Maidana et al. (2017), Van de Vijver et al. (2002 and Houk et al. (2017 and references therein) and Van de Vijver & Houk (2019).
For the typification of the new species, we chose to use the entire slide as the holotype following Art. 8.2 of the International Code for Botanical Nomenclature (Turland et al. 2018). Diatoms show a broad variability along their cell cycle making the choice for the entire population on the slide more obvious.

Ecology and distribution
The largest populations of A. chilensis were found in mosses growing on cliffs next to a waterfall dominated by several species of Humidophila, Diatomella balfouriana Grev. and Achnanthes muelleri C.F.W.Carlson emend. Van de Vijver & Goeyers. The latter species are all known to thrive mostly in aerophilic, moist environments ( Van de Vijver et al. 2002). Angusticopula chilensis was also found on several sub-Antarctic islands located in the southern Indian Ocean such as Iles Crozet (

Etymology
The specific epithet refers to the general outlook of the valves in scanning electron microscopy giving the impression, due to the many granules, of a cosmos with numerous stars.

Isotype
CAMPBELL ISLAND • same collection data as for holotype; slide at University of Antwerp, Belgium; PLP-368.

Ecology and distribution
Angusticopula cosmica sp. nov. was described from a Racopilum moss vegetation collected from a wet rock east of Moubray Hill. The sample was dominated by Frankophila dalevittii, a recently described endemic species for Campbell Island (

Etymology
The species is named in honour of our colleague and friend Dr Václav Houk (Institute of Botany, Czech Republic) to acknowledge him for his work on melosiroid diatoms.

Isotype
CAMPBELL ISLAND • same collection data as for holotype; slide at University of Antwerp, Belgium; PLP-369.   (Figs 67-68). Spines hollow but apparently chambered with small silica walls inside the spines (Fig. 68). Spines occasionally lacking or reduced to series of low silica outgrowths (Fig. 69). Central area clearly raised, bordered by a narrow flat valve face edge, covered by an irregular pattern of small, rounded to slit-like areolae (Figs 67, 68, arrows). Striation pattern not detectable in the areolae. Internally, a few sessile rimoportula visible close to the mantle edge (Figs 71, 72, arrows), irregularly scattered. One rimoportula present near the valve center on the valve face (Figs 70,73,arrow). Internal valve surface covered by numerous small areolae (Fig. 73).

Ecology and distribution
Ferocia houkiana sp. nov. is a frequently observed species on Campbell Island. It was found in several moss samples on rocks and next to waterfalls, usually dominated by species belonging to the genera Pinnularia, Pinnunavis and Eunotia. The type sample is dominated by the new species of Ferocia and is accompanied by Pinnularia borealis s. lat. Ehrenb. which points to a drier character of the environment, often influenced by sea spray (Van de Vijver et al. 2002

Description
Light microscopy  To date, frustules in girdle view not observed in LM. Valves strongly silicified, rounded with a thick mantle and clearly convex, weakly domed valve face. Valve dimensions (n = 10): valve diameter 9-18.5 μm. Central area formed by a large, smooth, hyaline zone, 6-11 μm, ca 60% of the total valve diameter, surrounded by a striated, irregularly bordered marginal zone. Thick spines, scattered in the marginal zone distinctly present. Marginal striae discernible in LM, 30-33 in 10 μm. Visible tube processes lacking.  Frustules discoid with very heavily silicified valves, visible as a thick mantle (Figs 116,(123)(124). Frustule height approx. 11 μm. Cingulum comprising at least 4, rather narrow, very thin, non-perforated, open, ligulate copulae (Figs 116, 120, double arrow). Pars interior clearly fimbriate (Figs 116,117,arrows) giving the impression of perforated copulae. Valve face with large, flat hyaline, almost rounded central area, free of areolae, but with a dense covering of small silica granules (Figs 119-121). Marginal zone distinctly sloping towards the mantle with a very narrow flat zone immediately next to the valve face/mantle junction. Regular striation pattern present on sloping marginal zone. Irregular marginal ring of large, acute, solid spines present (Figs 120-122) close to the hyaline central area, placed between the areolae. Irregular pattern of small granules (Fig. 122) extending between the spines, though never reaching the mantle. Striae composed of small, strictly rounded areolae. Areolae externally covered by perforated occlusions (Figs 118, 120). Openings of portulae not distinguishable from areolae even in SEM. Striae continuing over the valve face/mantle junction, interrupted by a thick marginal ridge running entirely around the mantle (Fig. 119), usually covered by girdle bands. Mantle edge rather narrow. Between marginal ridge and mantle edge, relatively deep groove present, bearing several rows of strictly rounded areolae.  Internally, areolae large, clearly rounded, each showing a distinct rota, organized in a regular striation pattern (Fig. 123). Mantle edge very broad, heavily silicified (Figs 123-124). Central area formed by a large hyaline, irregularly bordered, central zone (Fig. 123). Several perforated, thickened protuberances which we presume to be the inner openings of portulae are irregularly scattered between the areolae, never organized in a regular marginal ring (Fig. 123, arrows). Inner openings of these processes rounded, smaller than the areolae, weakly raised above the surface (Fig. 124).

Ecology and distribution
Arcanodiscus crawfordianus sp. nov. was described from a Racopilum moss vegetation collected from a wet rock east of Moubray Hill. The sample, in which also the newly described

Etymology
The specific epithet 'indistinctus' refers to the lack of any specific peculiarity of this species as a distinguishing feature.

Isotype
CAMPBELL ISLAND • same collection data as for holotype; slide at University of Antwerp, Belgium; PLP-371.

Scanning electron microscopy
Girdle composed of several, narrow, non-perforated copulae (Fig. 126). Pars interior of the copulae clearly fimbriate giving the impression of perforate bands (Fig. 126). Valve face with large, flat hyaline, almost rounded central area, lacking areolae or any other ornamentation (Figs 127-128). Marginal zone gently sloping towards the mantle, entirely covered with short slit-like to almost rounded areolae (the latter when clearly eroded), ca 45 in 10 μm, roughly organized in irregular striae. Areolae externally covered by small, weakly raised, individual occlusions (Fig. 127). No obvious tube process openings apparent. Striae continuing over the valve face/mantle junction, interrupted by a series of up to 3 thin, parallel ridges running entirely around the mantle (Fig. 129). Irregular silica thickenings, often connected to the first marginal ridge, present at the valve face/mantle junction (Fig. 128, arrows). Mantle areolae small, clearly rounded. Mantle edge very narrow, irregularly bordered (Fig. 129).

Ecology and distribution
Arcanodiscus indistinctus sp. nov. was described from a Racopilum moss vegetation collected from a wet rock east of Moubray Hill. The sample was dominated by by Frankophila dalevittii (Van de Vijver et al. 2020), Diatomella balfouriana and Diatomella colonialis. In the same sample, Arcanodiscus crawfordianus sp. nov. and Angusticopula cosmica sp. nov. were also observed. The presence in other samples needs to be confirmed with SEM observations.

Description
Light microscopy (Figs 97-115) Frustules broadly discoid, almost square to rectangular with broadly rounded edges and convex valve faces. Frustules often found attached to each other. Chains composed of more than 2 cells however never observed. Multiple discoid chloroplasts present. Girdle bands not discernible in LM. Valves rather strongly silicified, rounded with a thick mantle and convex, weakly domed valve face. Valve dimensions (n = 25): valve diameter 6-17.5 μm, frustule height (n = 10): 4.5-7.0 μm. Central area is a large, smooth, hyaline zone, 3.0-7.5 μm, 50-55% of the total valve diameter, surrounded by a more rugose, irregularly shaped, marginal zone where striae are difficult to distinguish. Visible processes lacking.
Valve face with large, flat hyaline, clearly rounded central area, lacking areolae and any other ornamentation (Figs 134-136). Marginal zone gently sloping towards the mantle, entirely covered with rounded to slit-like areolae, ca 45 in 10 μm, organized in irregular striae, the latter ca 40 in 10 μm. Areola occlusions externally not observed (Figs 134-135). External openings of portulae not distinguishable from areola openings. No silica thickenings, close to valve face/mantle junction (Fig. 135). Striae continuing over the valve face/mantle junction, usually interrupted by a series of up to 3 parallel ridges running entirely around the mantle (Figs 134-135). Parallel ridges, usually covered by girdle bands (Fig. 133), occasionally lacking and resulting in a broad, flat mantle (Fig. 136). Areolae on the mantle strictly rounded. Mantle edge indistinct. Internally, areolae rather large, each showing a clear rota, covered by very thin hymenes, organized in irregularly running striae (Fig. 138). Central area a large hyaline, irregularly bordered, central zone (Fig. 137). Large number of perforated, thickened protuberances visible, irregularly scattered between the areolae, never organized in a regular marginal ring (Fig. 137, arrows). Inner openings of these protuberances rounded, smaller than the areolae, lacking rota (Fig. 138).

Ecology and distribution
Arcanodiscus saundersianus sp. nov. was found in a sample collected from a dripping bluff limestone. The sample is entirely dominated by the new species of Arcanodiscus. Sub-dominant species include Achnanthes muelleri, Diatomella balfouriana and several species of Humidophila.

Discussion
The centric diatom flora of Campbell Island is represented by at least three different genera: Angusticopula, Ferocia, both belonging to the Melosirales, and Arcanodiscus that was placed in the order of the Arcanodiscales (Maidana et al. 2017). All species are characterized by the presence of numerous narrow, often unperforated girdle bands which is typical of these genera but entirely different from Melosira s. str. which has broad, perforated copulae . Additionally, the position and number of rimoportulae separate the three genera further from Melosira s. str. Table 1 gives an overview of the four genera and highlights the distinguishing features. Based on this table, it is possible to place each of the new species in one of the new genera. Characterizing features guiding these decisions include the presence/absence and position of rimoportulae, presence/absence of internal cells, the shape of the valve (domed, flat, sloping mantle or not), the presence/absence of marginal rings and the presence/absence of large, linking spines.
Both Ferocia and Angusticopula contain a large proportion of species that prefer aerial habitats ( Van de Vijver et al. 2017;Houk et al. 2017). As these habitats are likely to dry out occasionally, cells should protect their content from drying out. The presence of internal valves might be an important way to protect the cells against desiccation. The two genera possess these internal valves in many of their species. Melosira, considered to be a more aquatic genus (Round et al. 1990) is not known to present internal valves . Species of Arcanodiscus also seem not to present internal valves but still can be found in aerial habitats. It is, however, unclear why internal valves are lacking.
The description of the three new taxa of Arcanodiscus requires slight, but important changes to the original genus description (Maidana et al. 2017). Careful reanalysis of the original description of the type species and the accompanying illustrations shows that the girdle bands are open and not closed as stated in the genus description. Each new species of Arcanodiscus presents narrow, thin open girdle bands with clear ligulae (see Figs 116,132,arrows). Moreover, the girdle bands are not perforated, but in fact possess a fimbriate pars interior that could give the impression of being perforated. This is actually clearly visible on fig. 17 in Maidana et al. (2017). In the description of A. desmetianus (Van de Vijver & Houk 2019), a similar fimbriate pars interior was also observed although in the description, the copulae were considered being perforated. Other features that seem to be different from the original genus description is the apparent lack of portulae in A. indistinctus sp. nov., the presence of external, though open areolar coverings, the presence of spines in A. crawfordianus sp. nov. and the position of the portulae, being more irregular than organized in a submarginal ring as in A. crawfordianus sp. nov. and A. saundersianus sp. nov. Therefore, the original genus description by Maidana et al. (2017) needs to be emended.

Arcanodiscus Maidana & E.Morales in Maidana et al. (2017) emended Van de Vijver
Frustules discoid, solitary or in short chains. Valves with flat and solid hyaline central area and a thicker mantle, the latter with faint radial lines corresponding to the mantle tubular areolae. Striae continuous from valve face margin to mantle. Areolae disposed in a quincunx arrangement, internally covered by rotae. Gradual or abrupt transition between valve face and mantle. Mantle usually bearing several parallel undulations upon which the girdle elements rest. Rimoportulae and fultoportulae absent, but a reduced type of portula with an internal opening situated on a prominence and a simple external opening is often present, usually scattered between the areolae but occasionally forming a submarginal ring at the abvalvar portion of the mantle. Cingulum comprising numerous, thin, open, ligulate, non-perforated elements. Pars interior of the copulae clearly fimbriate, suggesting the copulae being perforated.
The genus Arcanodiscus shows some resemblance to two marine genera: Podosira Ehrenb. and Hyalodiscus Ehrenb. (Round et al. 1990). Podosira possesses rimoportulae scattered over the entire valve face whereas Hyalodiscus shows a ring of marginal rimoportulae. Maidana et al. (2017) already discussed the differences between Hyalodiscus, Podosira and Arcanodiscus, and based most of the discrimination on the presence/absence of these rimoportulae and the girdle structure. Hyalodiscus shows bullulate areolae, a feature however never observed in any of the species of Arcanodiscus known at present, and the girdle bands in the latter are clearly closed, and not open as in Arcanodiscus, confirming the discrimination between Hyalodiscus and Arcanodiscus. Podosira on the other hand has open girdle bands, similar to Arcanodiscus, non-bullulate areolae (as in Arcanodiscus) and often a flat valve face (Round et al. 1990). A better structural analysis of the portulae and rimoportulae in these three genera should also shed more light on the possible relationship between them, although this is outside the scope of the present paper. Therefore, an emendation of the order and family description of Arcanodiscus is inappropriate at the moment, as this may obscure the relationships between the three genera.
The description of three new species of Arcanodiscus brings the total number in this genus now to five, all of them occurring in the southern hemisphere and (for four of them) more specifically in the sub-Antarctic region. Only the generitype species, A. plattii Maidana & E.Morales, was described from southern Patagonia (Maidana et al. 2017). The three new species can be distinguished from the two previously described species of Arcanodiscus. Table 2 highlights the main morphological features of  (Maidana et al. 2017;Van de Vijver & Houk 2019). Arcanodiscus saundersianus sp. nov. also shows the highest stria (up to 40) and areola (up to 45) density, higher than any of the other four species.
The recently described genus Angusticopula contains seven species, slightly more than in either Ferocia or Angusticopula. Table 3 shows the features of all species of Angusticopula known so far. One of them, A. chilensis, was only known from the original drawing by Grunow and several LM illustrations in Houk et al. (2017: pl. 63, figs 1-5). A search for original, unmounted material remained without result (A. Igersheim, Grunow collection, Vienna). The population found on Campbell Island (and also similar populations on the Prince Edward Islands and Iles Kerguelen; Van de Vijver, unpubl. res.) shows a very high resemblance to the type specimens as depicted in Houk et al. (2017). The presence of the rimoportula on the margin, clearly visible in LM, is very obvious and not observed in any other species of Angusticopula. The most similar species might be A. dickiei, but the latter possesses rimoportulae that are located close to the mantle edge and not on the valve face/mantle junction as is the case in A. chilensis. Angusticopula chilensis was described from Chile although neither the slide (Houk et al. 2017: pl. 63, fig. 7) nor the drawing in Van Heurck (1885: pl. 90, fig. 13) provide a more detailed indication of the correct locality. The presence of a diatom described from Chile on Campbell Island is, however, not surprising given the presence of more species originally described from southern South America on the island such as Adlafia tenuis Van    The final species, Ferocia houkiana sp. nov., shows features of both the genera Angusticopula and Ferocia such as the narrow copulae and the scattered rimoportulae ; Van de Vijver et al. 2017). The presence of the large central spines and the structure of the areolae, especially visible on the valve interior show more resemblance with Ferocia than with Angusticopula and therefore the species is placed within the former genus. The species is, however, unique within the genus Ferocia in having a distinct central ring of linking spines (  (2018) was observed whereas the genera Stephanodiscus, Lindavia, Pantocsiella and Cyclostephanos are entirely absent. Possible records of species belonging to these genera (or formerly placed in Cyclotella) should be considered as erroneous identifications or the result of contamination or force-fitting (Kellogg & Kellogg 2002). The almost complete absence of members of the latter five genera is most likely the result of the quasi-total absence of freshwater diatom plankton in Antarctica, a phenomenon that has up to now not been fully explained (Jones 1996;Van de Vijver & Beyens 1999). On the other hand, the melosiroid diatom flora seems to be overrepresented with 12 species, although only species belongs to Melosira s. str. The genera Ferocia, Angusticopula and Arcanodiscus are most likely composed of species typical of more aerophilic conditions influenced by seaspray and animals such as living on mosses, in soils exposed in cliffs, near waterfalls or near bird colonies (Cremer et al. 2011;Van de Vijver et al. 2017;Van de Vijver & Houk 2019). The observation of the five new species also highlights the diversity within the sub-Antarctic non-marine diatoms and, despite the taxonomic efforts of the past 15 years, the gap in our knowledge and the need of a continuous effort in documenting the biodiversity of these unique flora.