The diatom genus Orthoseira Thwaites (Orthoseiraceae, Bacillariophyta) was originally described in 1848 based on Melosira americana Kütz. (Kützing 1844). In its current concept, the genus encompasses a group of mostly aerophilous non-pennate diatoms with radial symmetry, a flat valve face and several distinct central tube-like pores, called carinoportulae (Crawford 1981). Following the latest molecular studies, revealing a close phylogenetic relationship with the ‘multipolar’ diatoms, representatives of the genus Orthoseira are now placed in the order Orthoseirales, class Mediophyceae (Gargas et al. 2018). Apart from the shared feature of the carinoportulae, members of the genus Orthoseira are characterized by quite some variability in their morphological features (Houk 1993; Houk et al. 2017). Moreover, a discussion of the taxonomic history of the genus Orthoseira indicated the unclear taxonomic identity of the genus (Danz et al. 2022) since the designated typus generis, M. americana, entirely lacks the presence of carinoportulae, a feature not even included in the genus description. Danz et al. (2022) recommended that the name Orthoseira should be restricted only for M. americana. A revision of all known Orthoseira species is currently being undertaken (Kociolek et al. unpubl. res.), taking also into account three genera that were described by Ehrenberg in 1848, several months after the description of the genus Orthoseira by Thwaites (1848) (Ehrenberg 1848), these three genera all possessing carinoportulae (i.e. Liparogyra Ehrenb., Porocyclia Ehrenb., and Stephanosira Ehrenb.). Together with the revision of the already described Orthoseira species, the analysis of unidentified populations of possible Orthoseira species worldwide is another crucial step towards a better understanding of this group of species. This has led to a steep increase in the number of Orthoseira species during the past years. According to AlgaeBase, there are currently 34 accepted names (species and formae included) in Orthoseira (Guiry and Guiry 2023). The past years, species have been described from the sub-Antarctic region, Argentina, the United States, Greenland, Switzerland, South East Asia, Easter Island, and China (Van de Vijver and Kopalová 2008; Lowe et al. 2013; Guerrero et al. 2018; Kociolek et al. 2021; Danz and Kociolek 2022; Rybak et al. 2022; Goeyers et al. 2023; Peszek et al. 2023).

The genus Orthoseira is generally known to be typically aerophilous, usually observed in samples collected from wet rocks, the spray zone of waterfalls, moss carpets on tree trunks, lava caves, and soils (Round et al. 1990; Lowe et al. 2013; Houk et al. 2017). The genus does not seem to be restricted to a particular climate zone as species of Orthoseira have been reported from the tropics to the polar regions (Van de Vijver et al. 2004; Van de Vijver and Kopalová 2008; Lowe et al. 2013; Houk et al. 2017; Danz and Kociolek 2022; Goeyers et al. 2023). Despite this worldwide genus distribution, most of the Orthoseira species seem to have a rather restricted biogeographic distribution (Van de Vijver and Kopalová 2008; Lowe et al. 2013), even being part of an endemic diatom flora (Kociolek et al. 2021; Danz and Kociolek 2022; Rybak et al. 2022). On the other hand, there are still Orthoseira species that are often considered cosmopolitan, such as O. roeseana (Rabenh.) Pfitzer, described originally as Melosira roeseana (Rabenhorst 1853), and transferred to the genus Orthoseira in 1871 (Pfitzer 1871). One of the reasons of this presumable cosmopolitan occurrence is the force-fitting of local populations into the name O. roeseana as the morphology of this species was poorly known, and only recently studied in detail based on the analysis of its type material (Kochman-Kędziora et al. 2023).

As a result, the name O. roeseana is not only reported from all over the world, the morphology of O. roeseana has also been broadening during all these decades. The recent analysis of the type material of both Orthoseira (Melosira) roeseana and O. spinosa W.Smith (Smith 1855) proved that most O. roeseana populations in fact represent independent, often undescribed species (Kochman-Kędziora et al. 2023), underestimating the diversity of the genus Orthoseira, as confirmed by the recent description of for instance O. tatrica Houk, Klee & H.Tanaka (Houk et al. 2017), O. groenlandica Goeyers, Kochman-Kędziora & Van de Vijver (Goeyers et al. 2023), and O. helvetica Peszek, C.T.Robinson, Rybak & Kawecka (Peszek et al. 2023), all showing some similarity with O. roeseana.

During a survey of the aerophilic diatom flora in Papua New Guinea (Karkar Island, Boisa Island), four morphologically distinct Orthoseira taxa have been observed. Two taxa could be identified as O. hawaiiensis Danz & Kociolek (Danz and Kociolek 2022) and O. tropica (Krasske) Metzeltin & Lange-Bert. (Metzeltin and Lange-Bertalot 2007), whereas the two other taxa could not be identified using the currently available literature. Following light (LM) and scanning electron (SEM) microscopy observations, both unknown taxa are described as new species: Orthoseira iserentantii sp. nov. and Orthoseira papuensis sp. nov. Their morphology is illustrated and discussed in comparison with all known Orthoseira species worldwide.

During a survey of the aerophilic diatom flora in 1987 and 1989 in Papua New Guinea (Mont Wilhelm, Karkar Island, Boisa Island), more than 100 samples were collected from mosses and liverworts on fairly dry to humid, aerial substrates such as tree trunks and roots of bushes and trees. A preliminary survey of the samples resulted in a selection of four samples containing large populations of Orthoseira taxa:

A subsample of each of the selected materials was prepared for LM and SEM observations following the method described in van der Werff (1955). Small volumes of each subsample were cleaned by adding 37% H₂O₂ and subsequently heating to 80°C for about 1–2 h, after which the reaction was completed by adding saturated KMnO₄. Following digestion and centrifugation (three times 10 minutes at 4500× rpm), the cleaned diatom material was diluted with distilled water to avoid excessive concentrations of diatom valves on a slide and mounted in Naphrax®. Slides were analysed using an Olympus BX53 microscope at ×1000 magnification (UPLanFL N 100× objective, N.A. 1.30), equipped with Differential Interference Contrast (Nomarski) optics and the Olympus UC30 Imaging System. For each taxon, the number of specimens, measured at random on the type slide, is indicated (n = X).

For SEM, parts of the oxidized suspensions were filtered through a 5-µm Isopore™ polycarbonate membrane filter (Merck Millipore). Filters were air-dried and pieces were subsequently affixed to aluminium stubs. The latter were sputter-coated with a platinum layer of at least 15 nm and studied using a JEOL-JSM-7100F field emission scanning electron microscope operated at 2 kV and 4 mm working distance (Meise Botanic Garden, Belgium). Slides, samples and stubs analysed in this study are stored at the BR collection (Meise Botanic Garden, Belgium). Plates were prepared using Photoshop CS5.

Terminology used for the description of the various structures of the siliceous cell wall is based on Ross et al. (1979, areola structure), Crawford (1981, Orthoseira genus features), and Houk et al. (2017, Orthoseira genus features). For the taxonomic treatment, the following papers were consulted: Van de Vijver and Kopalová (2008), Lowe et al. (2013), Houk et al. (2017), Guerrero et al. (2018), Kociolek et al. (2021), Danz and Kociolek (2022), Rybak et al. (2022), Goeyers et al. (2023), Kochman-Kędziora et al. (2023), and Peszek et al. (2023).

For 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). An appropriate image is linked to the designated type by stating “Fig. X illustrates the type”, ensuring that the identity of the species can be fixed.

The observations of the Orthoseira populations in the samples from Papua New Guinea allowed the separation of four different Orthoseira taxa. Two populations could be identified as taxa that were previously described: Orthoseira hawaiiensis and O. tropica. The two other taxa are described as new following a thorough comparison with all previously recognised species: Orthoseira iserentantii sp. nov. and O. papuensis sp. nov. The morphology of all four species is discussed and compared to known species worldwide.

The morphology of all four species corresponds to the description of the genus Orthoseira as defined by Crawford (1981), based on the presence of the typical carinoportulae, the broad perforated copulae and the deep mantle. The four species each present a unique combination of features allowing the ability to separate them in both LM and SEM. Differences between the four species include the presence of internal caverns visible on the valve interior as marginal porefields (O. hawaiiensis), the presence of small slits between the areolae in the valve interior (O. hawaiiensis, O. tropica, and O. iserentantii), the number of rows of perforations on the copulae (maximum 2 in O. tropica, multiple in the other three), the presence of plate-like linking structures (O. tropica and O. iserentantii), the presence of marginal spines (O. hawaiiensis and O. papuensis), and the external structure of the carinoportulae (with plate-like coverings in O. hawaiiensis and O. papuensis, open in O. tropica and O. iserentantii). Whether these differences will allow the separation of groups within Orthoseira sensu Crawford (1981) can only be confirmed after a thorough cladistic analysis, which is out of the scope of the present paper.

Two species were previously described from Hawaii (O. hawaiiensis, Danz and Kociolek 2022) and South America (O. tropica, Krasske 1948).

Orthoseira hawaiiensis has up to now only been observed on Maui Island (Hawaii, Danz and Kociolek 2022). Comparison between the type population and Papua New Guinea population in the present paper, revealed no clear morphological differences, apart from the observation of some specimens with only two carinoportulae (whereas Danz and Kociolek (2022) strictly mention 3 carinoportulae). Both populations have externally covered carinoportulae, areas on the valve face with denser ridge aggregations, organised in a circular pattern, short, robust marginal spines, internal caverns, multiareolate broad copulae, and internal slits between the carinoportulae, justifying the proposed conspecificity (Danz and Kociolek 2022). The maximum valve diameter in Danz and Kociolek (2022) is more than double that of the observed diameter in the Papua New Guinea population. However, as the diameter range of the Papua New Guinea population is entirely fitting within the Hawaiian range, this difference should be neglected. Interestingly, O. hawaiiensis was described from mosses growing in lava tubes, whereas the Papua New Guinea population was found on moist liverworts growing on a tree trunk.

Lange-Bertalot et al. (1996) illustrated Melosira roeseana var. tropica Krasske with two (partial) LM images (Lange-Bertalot et al. 1996: plate 53, figs 5, 6). At present, this is the only representation of the type population of this species that is currently available. Rybak et al. (2022) observed and illustrated a large population of O. tropica on Palambak Island (Indonesia). Based on its morphology, the Papua New Guinea population in the present paper shows a large similarity with the Palambak population, but also presents several morphological differences. Similarities include the often spiralling plate-like structures, the broad girdle bands with maximum 2 rows of perforations, and the siliceous outgrows on the plate-like structures (see Fig. 2G). In the Palambak population, at least 2, but more often 3–4 rows of areolae are present between the plate-like linking spines similar to the Papua New Guinea population, where also 1–4 rows of areolae can be seen (Rybak et al. 2022: figs 34–36, and our Fig. 2H, J). On the other hand, the hyaline central area in the Palambak population is much larger than the central area in the Papua New Guinea population; in the latter, the areolae is almost touching the carinoportulae (see Rybak et al. 2022: figs 34–36 compared with our Fig. 2J, K). Whether the latter difference is sufficient to separate both populations is unclear: firstly, a better knowledge of the morphological variability of the type of O. tropica is necessary; and secondly, more populations identified as O. tropica should be analysed, to verify how variable this feature is.

The two other species are described as new, as they present clear differences with all known species. Orthoseira iserentantii shows a high degree of similarity with Orthoseira radiata M.Rybak, Glushchenko & Kulikovskiy, described from Palambak Island in Indonesia (Rybak et al. 2022). However, analysis of the valves illustrated in Rybak et al. (2022: figs 101–106) shows that most likely fig. 102, taken from a specimen from Rapa Nui (Easter Island), illustrates a valve that does not belong to O. radiata, based on significant differences in mantle striae, hyaline central area and the siliceous ridges on the valve face. Both O. iserentantii and O. radiata have overlapping valve dimensions, although O. iserentantii has a slightly higher mantle areola density (ca 24 vs 20–22 in O. radiata). The two species differ in the arrangement of the raised siliceous ridges on the valve mantle, the structure of the copulae, the striation pattern on the mantle and the internal valve view. Orthoseira iserentantii presents an irregular network of raised siliceous ridges in the central area, even between the carinoportulae. Part of these ridges is connected to the plate-like linking structures. Similar structures can also be found in Orthoseira radiata, although in this species the ridges are shorter and more irregularly shaped ridges (Rybak et al. 2022: figs 101, 104). Additionally, the mantle in O. iserentantii, besides having a higher areola density, is composed of areolae with a cribrate internal structure that is even visible in the external view. In O. radiata, this cribrate structure does not seem to be present, based on Rybak et al. (2022: fig. 105). The copulae in O. radiata appear to be very broad, bearing multiple rows of small areolae, whereas in O. iserentantii the copulae are narrower with only two or a few rows of large areolae. Finally, both species can be separated based on their internal structure. The areolae in O. radiata are occluded by a velum, whereas in O. iserentantii they have a cribrate structure (although it is possible that the vela have been eroded, even though this was not the case in the other Orthoseira species analysed in this study that have been prepared simultaneously in the same way). Additional features such as the slit-like openings between striae and the delicate ornamentation of the carinoportulae in O. iserentantii, are absent in O. radiata. Given that Rybak et al. (2022: fig. 102) probably does not belong to O. radiata and based on the statement that O. radiata is characterized by a high morphological variability (Mateusz Rybak pers. comm.), it is possible that fig. 102 represents O. iserentantii, indicating that the samples analysed by Rybak et al. (2022) most likely contain more species, including O. iserentantii, than actually reported. Further analysis of the Palambak and Rapa Nui samples in Rybak et al. (2022) will be necessary to confirm the species identity of the specimens presented by Rybak et al. (2022: fig. 102).

Orthoseira papuensis sp. nov. can be confused with O. cylindrica M.Rybak, Glushchenko & Kulikovskiy (Rybak et al. 2022), especially when using only light microscopy observations. Both species overlap in valve diameter (5.8–26.0 µm in O. cylindrica vs 8.0–21.5 in O. papuensis), but have a different stria density (22–24 in O. cylindrica vs 16–22 in O. papuensis). The ultrastructure of both species, as revealed by the SEM analysis shows that both species are quite distinct. The valve face of O. cylindrica is ornamented with radiating striae composed of round, rimmed areolae (Rybak et al. 2022: figs 49, 50, 52), contrarily to O. papuensis, in which the valve surface is characterised by a combination of rimmed areolae and an irregular pattern of small, irregularly long siliceous ridges that are present on the entire valve face, including between carinoportulae. Similar ridges are almost absent in O. cylindrica. Both species also differ in the arrangement of the pores on the girdle bands. Whereas on the girdle bands of O. cylindrica the pores are organised in rows (Rybak et al. 2022: fig. 54), the girdle bands in O. papuensis have a less organised pattern of pores (see Fig. 4M). Internally, O. cylindrica has areolae covered by well separated individual vela (Rybak et al. 2022: fig. 51), whereas in O. papuensis, the areolae are covered by an apparently continuous strip of fused vela running from valve margin to the carinoportulae (Fig. 4N).

The discovery of two new Orthoseira species on Papua New Guinea shows that there is still a large unknown aerophilic diatom flora present in remote places on Earth. Nevertheless, the observation of two other, previously described tropical Orthoseira species proves that there might be a pool of tropical aerophilic species present in environmentally similar habitats worldwide. The results also showed that due to lumping and force-fitting, the actual diversity of the genus Orthoseira is still poorly understood. Unpublished results from the sub-Antarctic regions showed that the reported, presumable cosmopolitan species in that region, in fact have to be described as new species, showing distinct differences with for instance O. roeseana s.s. A better understanding of the morphology of the different Orthoseira populations worldwide will most likely result in a better delimitation of all Orthoseira species, confirming the growing idea that even in aerophilic, seemingly cosmopolitan species, a distinct bioregionalisation is far more realistic.