Four new monoraphid diatom species (Bacillariophyta, Achnanthaceae) from the Maritime Antarctic Region

Four monoraphid taxa belonging to the genera Achnanthes, Psammothidium and Planothidium were found during the ongoing taxonomic revision of the freshwater and limno-terrestrial diatoms of the Maritime Antarctic region. The present paper describes these four taxa as new based on detailed light and scanning electron microscopy observations: Achnanthes kohleriana Kopalová, Zidarova & Van de Vijver sp. nov., Planothidium wetzelectorianum Kopalová, Zidarova & Van de Vijver sp. nov., Psammothidium confusoneglectum Kopalová, Zidarova & Van de Vijver sp. nov. and Psammothidium superpapilio Kopalová, Zidarova & Van de Vijver sp. nov. The morphology and ecology of all four taxa are discussed and the species are compared with morphologically similar taxa.


Introduction
The past 10 years, the non-marine diatom flora of the Maritime Antarctic Region has been undergoing a serious revision following a fine-grained taxonomy that has been widely accepted nowadays.Prior to this revision, only very sparse literature existed on the diatom composition of the Antarctic region and the reported diatom flora was based on a very broad species concept, making the use of this literature in biogeographical and biodiversity studies less appropriate due to the many inconsistencies in the diatom

Material and methods
During several austral summers (2004,2006,2008,2009,2013), material for diatom analysis has been sampled from James Ross Island and the South Shetland Islands (Livingston Island, Deception Island).In order to obtain a broad overview of the diversity and distribution of the diatom communities on the island, samples have been taken from different habitat types: freshwater lakes, seepage areas, wet rocks, rivers and mosses (ranging from aquatic to dry terrestrial).Samples were fixed in the field with 3% formaldehyde.Diatom samples for LM observation were prepared following the method described in Van der Werff (1955).Subsamples of the original material were oxidized using 37% H 2 O 2 and heating to 80°C for approximately 1h.The reaction was further completed by the addition of KMnO 4 .Following digestion and centrifugation (three times 10 minutes at 3700x g), the material free of organic matter was diluted with distilled water for sample mounting to avoid excessive concentrations of diatom valves on the slides.A subsample from the organic-free material was mounted in Naphrax® for diatoms community studies.The slides were analysed at BR using an Olympus BX53 microscope, equipped with Differential Interference Contrast (Nomarski).LM micrographs were taken using Olympus UC30 camera connected to the Cell Sense Standard program.For scanning electron microscopy (SEM), parts of the oxidized suspensions were filtered through polycarbonate membrane filters with a pore diameter of 1 µm, pieces of which were fixed on aluminium stubs after air-drying.The stubs were sputter-coated with a Gold-Palladium layer of 20 nm and studied in a ZEISS ULTRA SEM microscope at 3 kV.Terminology follows Hendey (1964), Barber & Haworth (1981), Round et al. (1990) and Lange-Bertalot (1993).
For the typification of the new species, we chose to use the entire slide as the holotype.In monoraphid diatoms, both valves of the same frustule show a different morphology, so two valves should be chosen to represent the holotype.Moreover, diatoms show a broad variability along their cell cycle making the choice for the entire population on the slide more obvious.

Ecology and distribution
So far Achnanthes kohleriana sp.nov.has been observed with certainty on several islands of the South Shetland archipelago (Livingston Island, Deception Island and King George Island).The largest population was found on Deception Island among wet mosses growing on a rock, located inland and far from the influence of sea sprays and with no nutrient input from sea birds or seals suggesting that the species is typically aerophilic.Other taxa present in the sample include Humidophila keiliorum Kopalová in Kopalová et al. (2015) 53).Distal fissures absent (Fig. 54).Raphe terminating immediately beyond the last stria (Fig. 54).Striae composed of 3-5 rows of small, rounded areolae (Figs 53,54).Near the axial area, each stria composed of usually 4-5 rows of areolae, towards the valve margin only 3 rows of areolae present in the stria (Fig. 54).Virgae clearly narrower than the striae (Fig. 53).Striae only shortly continuing onto the mantle (Fig. 53).Internally areolae covered by star-shaped hymenes (Fig. 55).Virgae clearly thickened and raised.Proximal raphe endings short, deflected (Fig. 55).Distal endings terminating onto small helictoglossae (Fig. 55).Rapheless valve (Figs 56-57): axial area weakly lanceolate.Striae always composed of 3 rows of small, rounded areolae (Fig. 56).Towards the axial area, striae becoming narrower (Fig. 56).Virgae usually as broad or slightly narrower than the striae (Fig. 56).Asymmetric central area clearly observable.Internally areolae covered by hymenes (Fig. 57).Virgae clearly thickened (Fig. 57).

Ecology and distribution
Planothidium wetzelectorianum sp.nov.was only found on James Ross Island in the epilithon and epipelon of Monolith Lake on the Ulu Peninsula.This relatively large lake has an almost circumneutral pH (7.2), a rather lower conductivity value (120 µS/cm) and low nutrient and sulphate values.mantle.Raphe branches almost straight.Proximal raphe endings straight, drop-like expanded (Fig. 83).Distal endings short, weakly drop-like expanded.Rapheless valve (Figs 84-85): central area slightly depressed, forming a weak pseudoraphe (Fig. 84).Striae uniseriate composed of small, rounded to rectangular and transapically elongated areolae (Fig. 84).Slit-like areolae present near the valve margins and onto the mantle (Fig. 84).In the central area, several slit-like areolae visible near the valve margin.Internally areolae transapically elongated, covered by individual perforated hymenes (Fig. 85).Virgae slightly raised.Small silica bars separating the areolae (Fig. 85).

Ecology and distribution
This new species was up to now only observed on Livingston Island.The largest population was found living in mosses submerged in a large lake showing a circumneutral pH (7.3) and a low conductivity level (< 100 µS/cm).The sample was dominated by several Psammothidium taxa such as P. subatomoides (Hust.)Bukht.& Round (Bukhtiyarova & Round 1996)

Etymology
The specific epithet refers to the similarity with P. papilio and the larger valve dimensions.

Ecology and distribution
This new species was up to now only observed with certainty on Livingston Island.The largest population was found living in a terrestrial moss vegetation, at the edge of a shallow lake.The sampling site was clearly influenced by animals with remains of penguins present on the mosses.The sample was dominated by several Psammothidium taxa such as P. germainii (Manguin) Sabbe (Sabbe et al. 2003)

Discussion
Although the Antarctic Region is characterized by a large number of Achnanthes, Psammothidium and Planothidium taxa, the new survey nevertheless resulted in the description of four new taxa.When separating these new taxa from all similar taxa known so far worldwide, the entire combination of features of each taxon has been taken into account to justify their status as independent taxa.Valve outline, raphe structure and striation pattern and structure are the main features that are investigated and compared between species.
Recently Tofilovska et al. (2014) revised the type material of A. coarctata (Bréb.)Grunow in Cleve & Grunow (Cleve & Grunow 1880) and some of its varieties.Based on their and our observations, it is clear that Achnanthes kohleriana sp.nov.belongs to the group of species around A. coarctata: both taxa present a similar position of the rapheless sternum, a comparable orbiculus, a similar raphe structure and similar cribrate areolae.Achnanthes kohleriana sp.nov.can however be easily separated from A. coarctata, often found in aerial habitats in Antarctica (Kopalová et al. 2012), by its valve outline, lacking the prominent constriction in the valve middle, typical for A. coarctata, by the marginal ridge which is less developed in A. kohleriana, and the lower stria density (10-11 in A. kohleriana sp. nov., 12-14 in A. coarctata).Achnanthes sinaensis (Hust.)Levkov et al. (Tofilovska et al. 2014) shows a comparable valve outline, although the valve middle is slightly more tumid (Tofilovska et al. 2014) and the apices are weakly protracted and more truncated and less broadly rounded, compared to A. kohleriana sp.nov.The marginal ridge in A. sinaensis is more developed (see for instance Tofilovska et al. 2014, fig. 13c).Moreover, the striae on the mantle in the latter are uniseriate, contrary to A. kohleriana sp.nov.which has biseriate striae on the mantle.Most other Achnanthes s.s.taxa showing a similar valve outline to A. kohleriana sp.nov., are usually typically marine or brackish taxa such as varieties of A. brevipes C.Agardh (Agardh 1824) or A. parvula Kütz.(Kützing 1844).Achnanthes parvula has smaller valves with a length not exceeding 30 µm and coarser areolae (Witkowski et al. 2000).Achnanthes parvula also shows a more laterally located rapheless sternum (McIntire & Reimer 1974, figs 4a, b), compared to A. kohleriana sp.nov.where the sternum is situated close to the margin.Achnanthes brevipes var.intermedia (Kütz.)P.T.Cleve (Cleve 1895) has a similar valve outline but a slightly coarser striation usually having 3 areolae per stria with much coarser areolae.Additionally, A. brevipes var.intermedia has square-shaped areolae and not rounded as is the case in A. kohleriana sp.nov., Fig. 22 (Toyoda & Williams 2004).Achnanthes subsessilis Kütz.(Kützing 1833) has a similar valve outline, but narrower valves with a width of around 7 μm (Hendey 1951), and not above 9 μm as in A. kohleriana sp.nov.Internally the central area is clearly panduriform and elevated from the rest of the valve (Blunn & Evans 1981, fig. 4).Several (poorly known) varieties of A. subsessilis also show some resemblance.Achnanthes subsessilis var.angusta Cleve & Möller (Cleve & Möller 1878) has a very narrow central area (contrary to the relatively broad central area in A. kohleriana sp.nov.).Achnanthes subsessilis var.multiarticulata Kütz.(Kützing 1844), (illustrated by Cox 2006, figs 6, 7 from type slide BM 18469) has a different valve outline with more lanceolate valves with less radiate striae and much smaller central area, as well as a lateral position of the sternum.Moreover, all A. subsessilis varieties are only found in marine conditions whereas A. kohleriana sp.nov. is a typical terrestrial taxon.Of the known aerophilic Achnanthes species, a few share similar features to A. kohleriana sp.nov.Achnanthes prominula Levkov & Tofilovska (Tofilovska et al. 2014), a recently described species from Osogovo, Macedonia, differs in having lanceolate or elliptic-lanceolate valves with narrowly rounded apices (and not linear to linear-elliptic valves with rounded apices), and with slightly coarser striation of 12-13 striae in 10 µm with only 5 areolae per stria (Tofilovska et al. 2014), opposite to A. kohleriana sp.nov.where the number of the striae in 10 µm does not exceed 12. Several other Achnanthes s.s.taxa are present in the Antarctic Region such as A. muelleri Carlson (Carlson 1913) and A. taylorensis T.B.Kellogg et al. (Kellogg et al. 1980).Although the latter was described from the Antarctic Continent (Kellogg et al. 1980), it was recently observed on James Ross Island in the Maritime Antarctic Region (Kopalová et al. 2012, figs 5C, 5D).Achnanthes taylorensis has smaller valves (length 31-33 µm and width of 8.8-9.3 µm) with a more lanceolate outline.Achnanthes muelleri, described from South Georgia in (Carlson 1913), has a different valve outline with larger, more rhombiclanceolate valves with clearly convex margins, making confusion with A. kohleriana sp.nov.that shows more linear-elliptic valves less likely.
Planothidium wetzelectorianum sp.nov.can be confused with several small-celled Planothidium taxa that all lack the presence of a cavum or a sinus.Several of these species co-occur in the Maritime Antarctic Region.Planothidium renei (Lange-Bert.& Rol.Schmidt) Van de Vijver in Van de Vijver et al. (2002) has a similar valve outline but differs in having typically only two rows of areolae in both rapheless and raphe valve (Schmidt et al. 1990;Oppenheim 1994).Moreover, P. renei has elongated external distal fissures continuing beyond the last stria whereas P. wetzelectorianum sp.nov.has only short distal fissures.The other Antarctic species, P. quadripunctatum (Oppenheim) Sabbe in Sabbe et al. (2003) has typically four rows of small, equally sized areolae per stria separated by very small virgae whereas P. wetzelectorianum sp.nov.has 3-4 areolae per stria, separated by rather broad virgae, with the largest areolae in the outer rows and the smaller areolae in the inner rows.Planothidium werumianum Lange-Bert.& Bąk in Bąk & Lange-Bertalot (2014) is also quite similar but has a more elliptic valve outline and typically has only 2-3 rows of small rounded areolae, separated by very large virgae, both on the raphid and rapheless valve.The axial area is usually broader (Bąk & Lange-Bertalot 2014).Finally, Planothidium granum (Hohn & Hellerman) Lange-Bert.(Lange-Bertalot 1999) has a different valve outline (elliptic-lanceolate with slightly rostrate apices), narrow virgae separating striae with 4 rows of very small areolae on the rapheless valve and strongly hooked external distal fissures.
Psammothidium confusoneglectum sp.nov.belongs to a group of small Psammothidium species that are widespread in the Antarctic Region.Similar species include P. confusum (Krasske) Bukht.& Round (Bukhtiyarova & Round 1996), P. stauroneioides (Manguin) Van de Vijver & Beyens in Van de Vijver et al. (2002), P. papilio and P. abundans.Psammothidium confusum is the most similar based on valve outline but the latter has a very different rapheless valve morphology with striae composed of maximum three transapically elongated, slit-like areolae (the longest ones being near the margin) and the absence of a clear central area whereas P. confusoneglectum sp.nov.has a typical fascia bordered by a series of only one areola.The raphe valve in P. confusum has a rectangular central area contrary to the wedge-shaped central area in P. confusoneglectum sp.nov.(Van de Vijver et al. 2002, Le Cohu 2005).Psammothidium abundans, present in the same locality as P. confusoneglectum sp.nov.has an almost linear valve outline with broadly rounded, slightly protracted apices contrary to the lanceolate valve outline in P. confusoneglectum sp.nov.The striae in P. abundans are more or less parallel in both valves whereas they are clearly radiate in P. confusoneglectum sp.nov.(Van de Vijver et al. 2008).Psammothidium papilio, widespread in the Maritime Antarctic Region has a more elliptic valve outline with a typical marginal crest on the rapheless valve, which has never been observed in P. confusoneglectum sp.nov.Moreover, P. papilio has a larger, more rounded, never wedge-shaped central area on both valves and a much lower stria density (26-28 in 10 µm vs 33-36 in 10 µm in P. confusoneglectum sp.nov.).The last similar species, Psammothidium stauroneioides has a broad lanceolate valve outline, larger valve dimensions (width 4.5-6.5 µm vs 3.8-4.5 µm) and a large, lanceolate axial area on the rapheless valve.
Finally, Psammothidium superpapilio sp.nov.can be separated from P. papilio in having larger valve length (15-20 µm vs 9-12 in P. papilio) giving the valves a more slender outlook since the valve width in both species is the same.The valves of P. superpapilio sp.nov.have a more lanceolate outline with weakly protracted apices whereas in P. papilio valves usually are elliptic even in the longest valves lacking any protracted apices (Schmidt et al. 1990).The raphe valves of P. superpapilio sp.nov.have a larger, more rectangular to even wedge-shaped central area, often forming almost a fascia.Psammothidium papilio on the other hand has a raphe valve with a more rounded to rhomboid central area with several shortened striae, always bordering the central area.Although both species co-occur in some samples, a continuum between P. papilio and P. superpapilio sp.nov.has never been noticed, justifying the separation of the two species.Other Psammothidium species such as P. abundans or P. stauroneioides differ sufficiently in valve outline, striation pattern and dimensions to exclude conspecificity.

Conclusions
The description of these four new monoraphid species once more confirms the unique character of the Maritime Antarctic non-marine diatom flora.The new data improve our knowledge of the biodiversity and biogeography of the Antarctic diatoms.

Figs 14- 19 .
Figs 14-19.Achnanthes kohleriana Kopalová, Zidarova & Van de Vijver sp.nov.Scanning electron micrographs of the type population from Deception Island (South Shetland Islands, Antarctica).14. SEM external view of an entire raphe valve.15.SEM external detail of the areolae with the typical cribrate structure.16.SEM external view of the distal raphe ending.17.SEM external detail of the central area.18. SEM internal detail of the central area showing the hooked proximal raphe endings.19.SEM internal view of an entire raphe valve.Scale bars represent 10 µm for Figs 14 & 19, 1 µm for Fig. 15 and 5 µm for Figs 16-18.

Figs 25 -
Figs 25-57.Planothidium wetzelectorianum Kopalová, Zidarova & Van de Vijver sp.nov.Light and scanning electron micrographs of the type population in Monolith Lake (James Ross Island).25.LM view of a girdle view.26-38.LM views of raphe valves.39-52.LM views of rapheless valves.53.SEM external view of an entire raphe valve.54.SEM external detail of the areolae.55.SEM internal view of the raphe and the striae.Note the hymenes on the areolae.56.SEM external view of an entire rapheless valve.57.SEM internal view of an entire rapheless valve.Scale bars represent 10 µm for Figs 25-52, 1 µm for Figs 53-57.
The species is named after our friend and colleague Dr. Tyler Kohler (Charles University in Prague and University of Boulder, Colorado, USA) in recognition of his diatom ecology work in the Dry Valleys of the Antarctic Continent.