Sample materials were collected on 7 January 2021 at two sites on Bawean Island, Java Sea, Indonesia with local names Mangrove Hijau Daun – MHD (5°50'57.5"S, 112°43'3.6"E) and Gili Iyang (5°51'11.70"S, 112°38'51.10"E) (Fig. 1A). The diatom samples were taken from reef sand and coral rubble (pieces of dead coral) within the reef flat area which is located 1 km away from the mangrove swamp area or near the reef crest in MHD and around 500 m from the coast in Gili Iyang (Fig. 1D). The diatom samples from Sulawesi were taken from the intertidal area at Tanjung Perak of Tomini Gulf (1°18'2.974'′S, 120°37'37.009'′E) on 29 September 2022. They were collected from rocks in the coral reef area. The sampling point is part of Tomini Gulf, the largest gulf in Indonesia, which has 1,350 km of coastline, and the distance between the indentation mouth shorelines was 44.4 km (Fig. 1B).

Figure 1. 

Map of sampling sites A sites Gili Iyang Harbour and MHD on Bawean Island B site Tanjung Perak on Central Sulawesi C, D detailed location of sampling areas on Sulawesi Island and East Java E location of Sulawesi and Java Island on Indonesian archipelago.

The samples were treated with 10% HCl, removing carbonate minerals (ca. two days), and washed with deionized water thereafter. Samples then were treated by adding 37% H₂O₂ and boiling for 3–5 hours to remove organic matter (Górecka et al. 2021; Kryk et al. 2021). Finally, the boiled samples were rinsed five times with deionized water. Thereafter, the water suspension was pipetted onto coverslips, left at room temperature for 24 h until evaporation of the water, and mounted onto glass slides using Naphrax®. Light microscopy (LM) images were taken with a Zeiss Axio Scope A1 light microscope (Carl Zeiss, Jena, Germany), with a 100 × Plan Apochromatic oil immersion objective (n.a. = 1.40) equipped with a Canon EOS 500D camera (Li et al. 2015). For identification and terminology reference of diatoms, sources such as Witkowski et al. (2000) and Round et al. (1990) were used.

In addition, for scanning electron microscopy (SEM) imaging, a few drops of cleaned or raw material were placed on a Whatman (pore size 5 or 1 μm) Nuclepore® filter and rinsed twice with distilled water. Filters were mounted onto aluminum stubs and air-dried before coating with gold-palladium alloy using a sputter coater. SEM examination involved a Hitachi SU8020 from West Pomerania University of Technology in Szczecin (Poland), at the Faculty of Chemical Technology and Engineering, and a Hitachi SU8010 at the University of Rzeszów, Poland.

Paracatenula has unique features that make it recognisable, including its shape, striation in the mantle, girdle band, and a slit-like depression in the apical area, which we call a sulcus. The valve margin of the new genus has a dorsal convex and a ventral straight side, sharing similarities with Catenula. However, the valve apices in the new genus are protractedly deflected toward the ventral margin, which cannot be found in Catenula. The most similar genus which has the pointed valve ends that curve or bend downward is Halamphora, e.g., Halamphora normanii (Rabenh) (Levkov 2009) and H. montana (Krasske) (Levkov 2009), which have capitate valve apices in shape. In contrast, Paracatenula has rostrate valve apices (Table 1).

Striation in the valve mantle of Paracatenula is ornamented by single tiny uniseriate areolae. In other dorsiventral diatoms, such as Catenula and Catenulopsis, the mantle is featured by a row of elongated slits which are positioned perpendicularly to the surface of the valves (Fig. 2U). Paracatenula features an intricate unique cingulum structure with an open channelled and perforated band. Externally, on the dorsal side, the cingulum is ornamented with biseriate striae, creating a double row of fine, closely spaced lines. Internally, on the ventral side, it features a single row of elongated pores (Fig. 2S, T). The absence of such perforation patterns in Catenula highlights this distinctive morphological feature of Paracatenula. Several diatom genera with a perforated cingulum include Catenulopsis and Halamphora. Frustules of Catenulopsis are supported by several perforated girdle bands (Kryk et al. 2021). Another dorsiventral diatom which is composed of girdle bands perforated with a few rows of poroids is Halamphora catenulafalsa (Witkowski et al. 2016). However, the two unique types of cingula striation previously described are only found in Paracatenula. The perforated cingulum is common and found in centric diatoms, e.g., Thalassiosira and Stephanodiscus (Round et al. 1990).

The genus Paracatenula exhibits several distinctive characteristics that justify its classification within the Catenulaceae family (Table 1). The frustules of Paracatenula are rectangular in girdle view, have asymmetric dorsiventral valves in the apical plane, the raphe position is eccentric close to the ventral margin, valve apices are protracted and rostrate, there is striation in the mantle, and there is a plain valve face with transapical ribbing (Round et al. 1990; Levkov 2009; Pliński and Witkowski 2020). Additionally, the family Catenulaceae exhibits a variety of apical valve morphologies. For instance, Catenula has no protracted apices, while Amphora and Halamphora do have protracted, capitate apices that terminate on the dorsal mantle. Moreover, the protracted, rostrate valve apices of Paracatenula are rarely reported from other genera of Catenulaceae. Only C. pelagica has been reported to have slightly rostrate apices (Round et al. 1990).

The newly described genus Wallaceago differs from all established catenuloid genera in terms of its valve shape, stauros, and raphe. Wallaceago exhibits a distinctive valve morphology characterized by a semi-rhombic, dorsiventral shape with broadly rounded apices. The valves display a pronounced bilateral flattening along the dorsiventral axis while maintaining a semi-rhombic outline tapering towards the apices. This combination of dorsiventral compression and rounded apical terminations distinguishes Wallaceago from other closely related genera, e.g., Amphora, Halamphora, Catenula and Catenulopsis which possess valves with a semi-lanceolate, dorsiventral form (Barber and Haworth 1981; Round et al. 1990; Robert et al. 2019; Stepanek and Patrick Kociolek 2019; Kryk et al. 2021). So far, only Wallaceago exhibits these features. Moreover, striation in the mantle of Wallaceago is characterized by a distinctive pattern on the ventral side (70–80 striae per 10 µm) which is interrupted by a stauros. The stauros has been previously reported in C. adhaerens from Zimbros, Brazil but it is located in the central area of the valve face (Garcia and Talgatti 2011, p. 103, figs 16, 17). Note, in our opinion those described specimens are similar to C. decusa sp. nov. in this paper. The stauros is a distinctive valve feature found primarily in Catenula (Garcia and Talgatti 2011) and in the freshwater diatom, genus Stauroneis (Levkov et al. 2016). For distinguishing the fascia from the stauros, a key morphological feature in certain diatom taxa, refer to the comprehensive review by Cox (2012).

Wallaceago exhibits a distinctive biarcuate raphe system, characterised by both proximal and distal ends bent towards the ventral side of the valve. A unique feature of the raphe in this genus is the ornamentation along the slit, which resembles a framed profile. While Wallaceago shares some similarities in raphe morphology with the genus Catenulopsis, notable differences exist. In Catenulopsis, the raphe displays a more pronounced sinusoidal curvature, whereas in Wallaceago, the raphe tends to be less curved, with a straighter trajectory (Kryk et al. 2021).

The genus Wallaceago shows several distinctive characteristics that justify its classification within the Catenulaceae family. The frustule of Wallaceago has asymmetric dorsiventral valves, is rectangular in girdle view, the raphe position is eccentric close to the ventral margin, valve apices are never protracted, and there is striation in the mantle with a plain valve face with transapical ribbing (Round et al. 1990; Levkov 2009; Pliński and Witkowski 2020). The genus Wallaceago appears similar to the genus Catenulopsis in terms of the raphe position and direction of the raphe ends. Another shared characteristic between Wallaceago and Catenulopsis is the presence of striations on the valve mantle. However, a notable difference lies in the specific arrangement and nature of these striations. In Wallaceago, the striations are confined solely to the ventral mantle and consist of uniseriate areolae. In contrast, Catenulopsis exhibits striation patterns on both the dorsal and ventral mantles, with these striations appearing as single rows of puncta or pores. In addition, Catenulopsis exhibits areolate striations composed of columnar or lanceolate shapes similar to Amphora.

Paracatenula porostriata and C. javanica, exhibit intriguing similarities and differences. Both species possess a dorsiventral valve, a characteristic that is shared among catenuloid diatoms. Their transapical striae, a key feature in their structure, are barely resolvable when observed through the LM. However, a notable distinction lies in their apical forms. P. porostriata is characterized by protracted rostrate apices, a feature that is absent in C. javanica. As indicated in Table 2, there is a significant difference in the lengths of the two species. Specifically, the length of the newly described species, ranges from 10.1–26.6 µm. On the other hand, C. javanica, is shorter, with a length ranging from 9.7–15.4 µm (Table 2). Under LM observation, P. porostriata is easily misunderstood to be C. javanica (see Kryk et al. 2021; figs 53, 60) which has a dorsiventral shape with slightly protracted rostrate apices. Both species also have refractive proximal and distal raphe ends that appear as dots in a bright colour.

Ultrastructural observations using an SEM revealed distinct differences between the apices of the two species. Paracatenula porostriata exhibited apical pores that were conspicuously absent in C. javanica (Fig. 2O). A notable difference was observed in the transapical striae, with P. porostriata having fewer (14–22/10 µm) compared to C. javanica, which had 26–32/10 µm (Table 2). Furthermore, the raphe slit of P. porostriata was significantly longer, measuring 11.8 µm, in contrast to C. javanica, which measured only 6.8 µm. The cingulum of P. porostriata was characteried by biseriate striae externally and a single row of elongated pores internally. However, both shared a similarity in biarcuate shape of the internal raphe branches.

Based on observations of ultrastructures by using an SEM, Wallaceago porostriatus is characterised by an isosceles (semi-rhombic dorsiventral) triangular valve shape and a rectangular girdle shape. The established genus with a similar valve shape is Seminavis D.G. Mann, e.g., S. basilica. This species has a rhombic-lanceolate structure with truncated apices (Danielidis and Mann 2003). Distinct differences in the raphe structure differentiate the species S. basilica from W. porostriatus. Specifically, S. basilica is characterised by a straight raphe with a raphe fissure oriented towards the dorsal side. In contrast, W. porostriatus lacks such a raphe fissure. The raphe slit of W. porostriatus is notably short, measuring only 4.7 µm (Table 2). This is significantly shorter when compared to the similar genus Catenulopsis, which has a raphe slit length of 8.3 µm (Table 3; Kryk et al. 2021). Interestingly, the raphe slit length of W. porostriatus is nearly identical to that of Medlinella, which measures approximately 4.0 µm (Frankovich et al. 2016). The distal ends of the raphe in S. basilica and Medlinella exhibit a similarity to those of W. porostriatus, specifically, that they are curved towards the ventral side. However, the raphe of W. porostriatus is slightly arcuate in the middle, distinguishing it from the others. In general, raphe shape for W. porostriatus has slight curvatures whereas similar genera have raphe shapes that are more pronounced in bending or sinusoidal patterns along the ventral side. Another unique shape of the W. porostriatus raphe is attributed to the presence of a rabbet.

Among those species that have striation on the mantle, the type of striation differs. W. porostriatus has punctate striation, which means the striations appear as tiny, point-like dots. On the other hand, other species have slit-like striations, where the striations appear as small, elongated slits. The number of striations in the ventral area of the new species is higher, 70–80 per 10 µm, compared to M. amphoroidea (Tables 2, 3; Frankovich et al. 2016). Under LM, W. porostriatus and M. amphoroidea are identified as small diatoms, each with distinct valve lengths. The valve length of W. porostriatus ranges from 5.2–8.6 µm, while M. amphoroidea exhibits a slightly larger span of 7–13 μm. In contrast, Ca. catenulafalsa presents a longer valve length, measuring between 8.4–14.6 μm. Notably, the transapical groove in both M. amphoroidea and Ca. catenulafalsa is clearly visible under LM, a feature that is conspicuously absent in W. porostriatus.

Catenula boyanensis shows similarities and distinct morphological characteristics with C. javanica (Table 2). Its valve length and breadth are slightly less than C. javanica (Table 2). The valve shape of this species is semi-lanceolate dorsiventral, and it has a rectangular girdle shape. In C. javanica, the valve shape is strictly lanceolate with a broader dorsal side. The primary distinction lies in the external positioning of the raphe. In the case of C. javanica, it is situated nearly along the edge of the ventral valve face. Conversely, for C. boyanensis, the raphe is located on the valve face itself, significantly further from the margin. In C. boyanensis the path of the raphe slits is straight, with the distal raphe ending being straight externally but deflected to the dorsal side internally. The proximal raphe ending is straight and distant, both externally and internally. The length of the raphe slits is 9.1 µm, and they are positioned in the ventral area. On the other hand, the raphe distinctly exhibits an undulating pattern in C. javanica externally and shares a similar pattern internally. The raphe slit length is lower at 9.1 µm. The number of dorsal and ventral striae for C. boyanensis is lower compared to C. javanica (Table 2). Upon observation with LM, the valve shapes of both species appear to be similar.

The diatom species Catenula decusa shares similarities to C. adhaerens in terms of valve morphology and girdle structure. Both species exhibit a semi-lanceolate dorsiventral valve shape and a rectangular girdle form. However, notable distinctions can be observed between the two taxa. C. decusa is characterised by a shorter and slimmer frustule, with valves displaying a more pronounced lanceolate outline and a slender profile, in contrast to the broader, semi-lanceolate valves of C. adhaerens (length 6.8–12.4 μm, width 1.0–1.7 μm). Furthermore, the striation density is remarkably higher in C. decusa, with 35–40 striations per 10 μm, compared to the lower density of 25–35 striations per 10 μm observed in C. adhaerens. Additionally, the proximal ends of the raphe system in C. decusa exhibit a closer proximity 0.9 μm, while the distal ends are positioned nearer to the apices (0.8 μm). Another distinct difference is the thickening of the central area externally and internally in C. decusa with interrupted striation in the mantle, a feature absent in C. adhaerens. When observed under LM the central area seems lighter than other areas due to reflecting more light. Unlike C. adhaerens, C. decusa lacks helictogossae. In Kryk et al. (2021, p.10, figs 85, 93), the specimens that were incorrectly identified as C. adhaerens are C. decusa.

Catenula komodensis. The newly described species is distinct and does not resemble previously described catenuloids. This diminutive diatom species has some valves shaped as linear rhombic dorsiventral and others mostly semi-lanceolate dorsiventral. The second difference is, internally, the presence of a second silica deposit close to the apices on the ventral side that splits the striation. This species shares some similarities with two other species, W. porostriatus and Halamphora veneta, but C. komodensis is characterised by a smoothly arched dorsal margin, that distinguishes it from these related species. Valve morphometrics reveal a length ranging from 8.1–19.3 μm and a width spanning 1.1–2.1 μm, dimensions slightly larger than those of W. porostriatus (Table 2) yet approximately half the size of H. veneta (Levkov 2009). Notably, the proximal raphe endings are deflected towards the ventral side with a narrow gap between them, both externally and internally. The central nodule of C. komodensis extends towards the dorsal and ventral margins, manifesting itself as a thickening of the valve face similar to C. decusa. However, C. komodensis has two distinct silica deposits, situated in the central and apical areas, respectively. These deposits interrupt the striation patterns on both the dorsal and ventral aspects of the valve. Under LM, the valve shape is semi-lanceolate, and in smaller sizes, it appears lanceolate, and the silica thickening in the central and apical areas is clearly visible.

Catenula densestriata. Newly described C. densestriata is similar to Catenula adhaerens, Catenulopsis catenulafalsa, and Catenulopsis baweana. The significant difference is the number of dorsal mantle striae density in 10 μm which is 24–36 for C. adhaerens and 44–50 for the new species. Apical raphe ends are closer in proximity for Catenula densestriata at 0.3–1.2 μm in contrast to 1.5 μm for C. adhaerens. Internally, raphe slits of Catenulopsis are straight versus Catenula densestriata having raphe slits that are straight and apical raphe ends bent toward the dorsal side. One remarkable characteristic that distinguishes C. densestriata from other catenuloid species is its high striation density. Notably, the number of striations observed in this new species is approximately twice that of C. adhaerens (Sundbäck and Medlin 1986; Garcia and Talgatti 2011; Kryk et al. 2021), a significant characteristic of valve ornamentation.

Secondly, the distance between the external and internal proximal raphe endings in C. densestriata is nearly identical, measuring 1 μm and 0.9 μm, respectively. This feature is distinctly different from C. adhaerens, which exhibits twice the distance between its internal proximal endings and its external proximal endings (Kryk et al. 2021). Moreover, the distance between the distal raphe end and the apices is remarkably close in C. densestriata, 0.8 μm, with almost no difference between the external and internal morphologies. In contrast, C. adhaerens exhibits a greater distance of approximately 1.5 μm or twice that of the newly described species. Thirdly, the shape of the raphe slit’s path in C. densestriata exhibits an arcuate form, both internally and externally. Meanwhile, in C. adhaerens, the raphe slit is arcuate externally but straight internally (Kryk et al. 2021). Based on several distinguishing characteristics of C. densestriata, we believe that the specimen described as C. adhaerens in Kryk et al. (2021, p. 10, figs 84, 86) is Catenula densestriata.

Based on the description and explanation provided, the four new species C. boyanensis, C. decusa, C. komodensis, and C. densestriata exhibit the following general characteristics: a dorsiventral valve shape, a rectangular appearance when viewed from the girdle perspective, and slit-like areolae present on both the dorsal and ventral mantles. Additionally, they possess an eccentrically positioned raphe on the ventral side. Notably, in the case of C. boyanensis, distinct transapical grooves are observed. Furthermore, these species exhibit an open and unperforated cingulum structure.

Catenulopsis baweana sp. nov. (Ca. baweana) is the second species of the genus discovered and described thus far. The type of the genus is Catenulopsis catenulafalsa (Ca. catenulafalsa) described from the tidal flat habitats in Nosy Be Island in NW Madagascar (Kryk et al. 2021). Whereas there is a distinct overlap in valve length between the two taxa, firstly, the valve width is smaller in the newly described species, with 1.7–3.1 μm versus 3–4.1 μm in the species from Nosy Be. The most distinct differences are: the girdle bands pore density, which is 30–40 in 10 μm in Ca. catenulafalsa and 50–65 in 10 μm in Ca. baweana; the striated valve in Ca. catenulafalsa versus the plain valve face in the newly described species; and strongly dorsally bent raphe slits in the former species versus slightly bent in the latter species. Furthermore, Ca. baweana has a semi-lanceolate shape with an abrupt transition between the valve face and mantle. In contrast, Ca. catenulafalsa has a smoother transition between these structures (see Kryk et al. 2021, figs 27–32). Secondly, the raphe of the new species is situated significantly distant from the valve margin. However, the raphe of Ca. catenulafalsa is situated in a transition area. Consequently, the distal raphe endings clearly extend into the mantle (Kryk et al. 2021). Thirdly, both species possess biarcuate raphe slits; however, in the new species, the raphe is straight in the central region and exhibits curvature at the proximal and distal ends. This contrasts with the raphe in Ca. catenulafalsa, which tends to follow a sinusoidal path and is shorter in length (Table 3). Fourthly, unlike Ca. catenulafalsa, which is characterized by the presence of ribs on the ventral valve face, the new species exhibits a distinct absence of such structures. Fifthly, the dorsal and ventral mantles of Ca. catenulafalsa are striated by the irregular shape of long solitary areolae, while the newly described species has a lower number in 10 µm of dorsal striae (Table 3).

The authors have declared that no competing interests exist.

No ethical statement was reported.

This work was partly supported by the International Society for Diatom Research for the funding through Luc Ector Early Career Award 2023 and this publication is Co-financed by the Minister of Science the Republic of Poland under the Regional Excellence Initiative Program for 2024-2027 (RID/SP/0045/2024/01).

Conceptualization: AW, OML. Data curation: OML, AK. Formal analysis: AW, OML, MB, AK. Funding acquisition: MB, AW. Investigation: SA, YR, OML. Methodology: MR, LP, RJW. Project administration: Y. Resources: AW, MR, LP, RJW. Supervision: MB, JLP. Validation: AW, MB, MR, LP. Visualization: OML. Writing - original draft: AW, OML, AK. Writing - review and editing: JLP, MB.

Oktiyas Muzaky Luthfi https://orcid.org/0000-0002-9550-9381

Sulastri Arsad https://orcid.org/0000-0002-7322-7834

Adrian Kryk https://orcid.org/0000-0003-4959-4199

Yenny Risjani https://orcid.org/0000-0002-6191-5824

Yunianta https://orcid.org/0000-0002-9684-8131

Mateusz Rybak https://orcid.org/0000-0001-8998-9537

Łukasz Peszek https://orcid.org/0000-0002-9132-2210

Rafał J. Wróbel https://orcid.org/0000-0003-2593-0813

Janice L. Pappas https://orcid.org/0000-0002-1207-8388

Małgorzata Bąk https://orcid.org/0000-0001-6465-5047

Andrzej Witkowski https://orcid.org/0000-0003-1714-218X

All of the data that support the findings of this study are available in the main text.