Introduction

Raivavae (in Tahitian: Ra′ivāvae), part of the Austral Islands, also referred to as the Tupua’i Islands (South Pacific, Fig. 1), was first mentioned by Europeans in 1775, i.e., by the Spanish naval officer Tomas Gayagos, who was the commander onboard the frigate Aguila. Raivavae is a volcanic island (high island, erected between 10.6 and 5.4 Ma BP, Montaggioni 2015) with a large lagoon delineated by a coral-reef barrier with several motu (low coral islands), among which motu Vaiamanu (Fig. 1). Raivavae has a rocky shore and sheltered small bays with sandy beaches of coral sand. Ancestral marae are present on several parts of the island, witness to a rich culture and cultural past. The intertidal habitats of the lagoon were investigated in October 2018 to study monoraphid benthic diatoms. Several areas were sampled, in particular the fringe of Ruatara rock (“Rocher de l’Homme,” name referring to a Tahitian legend) (Fig. 1). Other sediments and seaweeds were also sampled on the North coast of Raivavae. The monoraphid diatoms in Raivavae include several genera (Table 1), upon which Achnanthidium Kützing, 1844, Amphicocconeis De Stefano & D Marino, 2003, Astartiella Witkowski, Lange-Bertalot & Metzeltin, in Moser et al. 1998, Cocconeis Ehrenberg, 1837, Madinithidium Desrosiers, Witkowski & Riaux-Gobin, in Desrosiers et al. 2014, Planothidium Round & Bukhtiyarova, 1996, Pseudachnanthidium Riaux-Gobin, in Riaux-Gobin and Witkowski 2015 and Scalariella Riaux-Gobin, in Riaux-Gobin et al. 2012 (Table 1). Among these genera, Cocconeis and Amphicocconeis had the most remarkable species diversity. Several new Amphicocconeis will be detailed elsewhere (C Riaux-Gobin pers. comm.). We here focus on a small linear-elliptical monoraphid taxon described as new: Cocconeis vaiamanuensis sp. nov. The new taxon is detailed with LM (light microscope) and SEM (scanning electron microscope) and compared with allied taxa. Some characters of the taxon, such as its valve shape and presence of small processes on the SV mantle (see description below), prompted us to establish a cladistic analysis, based on ultrastructure, giving some light about a possible connection among several genera such as: Psammothidium Bukhtiyarova & Round, 1996, Platessa Lange-Bertalot in Krammer & Lange-Bertalot, 2004, Lemnicola Round & Basson, 1997, Pauliella Round & Basson, 1997, Achnanthidium, Karayevia Round & Bukhtiyarova ex Round, 1998, Rossithidium Round & Bukhtiyarova, 1996 and Cocconeis. It can be first noted that, except for Cocconeis, the previously cited genera are mostly associated with freshwater, while the new taxon is a priori strictly marine. The morphological limits between some of the latter genera are particularly blurred, with incertitude about the particular position of several of them (see below). The use of SEM is often decisive in delineating taxa, and therefore strengthening the results of morphological cladistic analyses that permit to sort them. In the same manner, concerning the molecular phylogeny, without direct observation of cells under a microscope, it can be challenging to predict the identity of some diatoms as belonging to a particular genus, solely on their position in the molecular phylogeny (Frankovich et al. 2018).

Fig. 1
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Raivavae location in the Austral Archipelago (South Pacific), with location of Ruatara rock and motu Vaiamanu

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Table 1 Assemblage of Achnanthales from Raivavae (South Pacific)
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The genus Cocconeis is diverse in valve shape, ornamentation, and copula structure, allowing for several sub-groups to be identified, such as the Section Alveolatae De Stefano & OE Romero (De Stefano and Romero 2005) characterized by a bilayered complex SV, and the Section characterized by a marginal row of simple SV processes and complex RV striation (Riaux-Gobin et al. 2015), comprising Cocconeis peltoides Hustedt, 1939. The present study particularly seeks to provide insights into the phylogenetic and cladistic position of members of Cocconeis with or without processes. Such processes were named “poroids” in Tudesque et al. (2016) for some Platessa species, with the comment: “This type of ‘poroid’ requires further investigation because it could be reminiscent of an ancestral portula.” These structures were named “rimoportula” in Romero & Riaux-Gobin (2014) for Cocconeis pseudograta Hustedt, 1939. Here we prefer to use the term “simple process”, because these structures (that open externally via a tiny pore on the SV mantle and closed internally by a sort of polymorph domed hymen, see Riaux-Gobin et al. 2015) differ from true rimoportulae that are characterized by pair of lips in internal valve face, and simple pore or often more or less long tubes in external valve face, as illustrated in Round et al. (1990). Nevertheless, the difference in the complexity of these processes may be phenotypic differentiation among diatom Classes. SV processes were also observed in several Platessa (Romero 2016; Wetzel et al. 2017), Psammothidium (Potapova 2012; Blanco et al. 2017), Lemnicola (Shi et al. 2018), Rossithidium (Potapova 2012), Pauliella (Round and Basson 1997), Achnanthidium (Jüttner et al. 2020), and Karayevia (Spaulding and Edlund 2008).

On the other hand, recent increases in the number of phylogenetic analyses have provided important clues allowing us to propose genetic affiliations. A rbcL molecular analysis of a clone of Cocconeis cf. sigillata SZCZCH1200 (collected in Laoshan Shangquan coastal area, China) (see C. sigillata Riaux-Gobin & Al-Handal, in Riaux-Gobin et al. 2011a), a taxon similar to C. peltoides, with marginal SV processes, allowed for the illustration of the complex and diverse relationships of Cocconeis (currently called Cocconeis) with other genera. Our results add to those of other recent phylogenetic investigations (i.e., Nakov 2014; Kulikovskiy et al. 2016, 2019; Thomas et al. 2016; Witkowski et al. 2016; Shi et al. 2018).

A polyphyletic position of the genus Cocconeis is considered, and the term Achnanthales (Order Achnanthales Silva, 1962), up to now treated as homogenous (De Stefano and Marino 2003; Le Cohu 2005) is here reconsidered, as previously addressed in several other studies (i.e., Cox 2006; Cox and Williams 2006; Kulikovskiy et al. 2016; Davidovich et al. 2017).

Material and methods

Diatom materials

Marine materials (sediments, seaweeds, diverse scrapings) collected in October 2018 from Raivavae (Austral Archipelago) were observed with LM and SEM, and are here illustrated and discussed. A strain of Cocconeis cf. sigillata was isolated and cultured by Chunlian Li at SZCZ (Szczecin University) from a sand beach in Laoshan Shangquan coastal area, China (36.092 N; 120.469 E), isolate SZCZCH1200; 101 stored in the Szczecin Diatom Culture Collection (SZCZ), University of Szczecin, collectors: Yu Shu-xian, Wang Yin-chu, Wang Xiu-jing, and Witkowski Andrzej, sampled in June 2015, Witkowski Lab Voucher SZCZCH1200. The molecular marker analysis was performed (see below), along with LM and SEM examination at SZCZ, Szczecin University.

Sample preparation and examination

Materials (preserved in methanol) were washed with distilled water to remove salts (sedimentation method), treated with 30% H2O2 for 2 h at 70 °C to remove organic matter, rinsed several times in distilled water, alcohol-desiccated, and mounted on glass slides using Naphrax. Diatom slides were examined with a Zeiss Axiophot 200, with phase contrast and differential interference contrast (Nomarski interference contrast) optics and photographed with a Canon PowerShot G6 digital camera (CRIOBE-USR 3278, Perpignan, France). For SEM examination, drops of cleaned or raw material were filtered with a syringe-filter, through 1 μm Nuclepore® filters and rinsed twice with deionized (Milli-Q) water to remove salts. Filters were mounted onto aluminum stubs and air-dried before coating with gold-palladium alloy (EMSCOP SC 500 sputter coater) and examined with a Hitachi S–4500 SEM operated at 5 kV, calibrated with a Silicon grating TGX01 (C2M, Perpignan, France). Images kept in the authors’ collection and handled with Photoshop and Adobe-illustrator. Measurements were performed using SEM images (60 individuals of Cocconeis vaiamanuensis sp. nov. were measured).

Cladistic analysis method

A morphological cladistic analysis (ultrastructural characters) was applied to the taxa, including 16 characters (Table 2) and 34 taxa from 9 genera (Table 3). Taxa were selected following the accuracy of their SEM description, permitting or not to detail SV processes, RV transapical marginal poroids, and characteristics of the terminal raphe endings. The chosen characters consist of ultrastructural details allowing to define each of the nine genera included in the analysis: the shape of the valve, particular structures such as the mantle SV processes, valvocopula structure, stria pattern. For the outgroup, we selected a Planothidium (Planothidium juandenovense Riaux-Gobin & Witkowski in Riaux-Gobin et al. 2018). We used the software PAUP*4.0a165 (Swofford 2003). We ran a Bootstrap method with a heuristic search algorithm to search for the most parsimonious trees (100 bootstraps). Characters were analyzed as unordered and unweighted. The resulting 100 most parsimonious trees were then used to build a 50% Majority-rule consensus tree, which is presented in Figure 8.

Phylogenetic analysis method (DNA extraction, PCR, and molecular analysis)

Several clones, or strains, of Cocconeis and other genera, were cultured in SZCZ (Szczecin University), and molecular analyses were compared to data from GenBank®. Particularly, a clone of Cocconeis cf. sigillata SZCZCH1200 [see above details] was cultured and analyzed. Two strains of Triparma pacifica (Guillou & Chrétiennot-Dinet) Ichinomiya & Lopes dos Santos in Ichinomiya et al. (2016), p. 1432; Basionym: Bolidomonas pacifica Guillou & Chrétiennot-Dinet in Guillou et al. 1999, p. 371) were chosen as an outgroup. The final rbcL gene trees are available in Fig. 8. The full list of sequences used in phylogenetic analyses along with GenBank® accession numbers is available (Table 4).

DNA extraction using Chelex® 100 resin (Bio-Rad, cat. no. 142–2842-MSDS) followed the method described in Kryk et al. (2020). The molecular marker rbcL was amplified using primer and PCR protocol as described in Dąbek et al. (2017). The sequencing of the PCR products was performed using BigDye Terminator v.3.1 chemistry and ABI3730 xl sequencer by the oligo.pl DNA Sequencing Laboratory at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. For sequence assembling, BioEdit ver. 7.2.5 (Hall 1999) was used. For maximum likelihood (ML) estimation, phylogenetic tree was inferred with 1000 bootstrap replicates using rapid bootstrap analysis in RAxML v.8.1 (Stamatakis 2014). The best score for ML tree was chosen as the final tree, and bootstrap support values (bv) were added at nodes. For Bayesian Interference, the tree was carried out using MrBayes v3.2.7 (Huelsenbeck and Ronquist 2001). The data was partitioned as three different codon positions. Two Bayesian inference analyses each with four chains (one cold and three heated) were run using GTR + G + I model. During this procedure, the standard deviation of split frequencies was around 0.01 when it reached 150,000,000 generations, which were run per analysis with sampling every 1000th iteration, generating in total of 150, 000 samples. 25% of the samples were discarded, then the rest were used to get a majority rule consensus tree and obtain posterior probabilities for nodes.

Results

Marine Achnanthales assemblage from Raivavae (Table 1)

The microphytobenthos from the intertidal environments of Raivavae is highly diversified (in term of species number), particularly within the monoraphids (Table 1). We note the presence of several pantropical taxa, from which several were previously reported from the Indian Ocean. Some taxa belonging to Olifantiella Riaux-Gobin & Compère, 2009, and Amicula Witkowski, Lange-Bertalot & Metzeltin, 2000, were also present. Most monoraphids (Table 1) are present in several other places in the South Pacific. In contrast, Cocconeis vaiamanuensis sp. nov. may be a local endemic, since it has never been cited from elsewhere. During several sampling campaigns in the South Pacific, we noticed different productivity and species diversity of benthic diatoms, particularly concerning the monoraphids, as a function of the geomorphology of the site: atolls (carbonate environments) versus high volcanic islands (siliceous environments, see remarks in Riaux-Gobin et al. 2014). More than 37 monoraphid taxa were reported from Raivavae, upon which 25 Cocconeis (Table 1). Amphicocconeis, with several taxa, is detailed elsewhere.

Cocconeis vaiamanuensis Riaux-Gobin, Witkowski & Ector sp. nov. (Table 2; LM Fig. 2 a–p; SEM Figs. 3, 4, 5, 6)

Class: Bacillariophyceae Haeckel, 1878

Order: Achnanthales Silva, 1962

Table 2 Characters, state, and coding used in the Cladistic Analysis. *See definition in the text
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Fig. 2
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Cocconeis vaiamanuensis sp. nov. LM (Raivavae, Ruatara rock). a–j SV with linear-elliptical shape, round apices and regularly spaced striae; k RV with almost invisible striae; l–p complete frustules at different foci allowing for the RV and SV of each specimen to be delineated and visualized. Scale bars = 10 µm (Nomarski interference contrast); q Cocconeis finmarchica Grunow in Cleve & Grunow. Original drawings. Note the marginal hyaline area on both valves, and the RV fascia. Scale bar = 10 µm.

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Fig. 3
figure 3

Cocconeis vaiamanuensis sp. nov. SEM (Raivavae, Ruatara rock). a–b SV in external view. Slightly concave, with regularly spaced striae. Note the marginal crista marginalis (arrow). Cingulum composed of several large copulae (arrowhead); c dense marginal striation of the RV (arrowhead); d marginal raised portion of the SV virgae (multiple arrowheads); e–f position of the small aperture of each process on the SV mantle (twin arrowheads, twin arrows). Scale bars = 3 µm (c), 2 µm (a–b, d), 700 nm (e), 300 nm (f)

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Fig. 4
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Cocconeis vaiamanuensis sp. nov. SEM (Raivavae, Ruatara rock). a SV in internal view. Regular arrangement of striae composed of small round areolae with convex hymenes. Areolae near the sternum less dense than on the margin (arrowhead); a narrow and straight SV sternum; b SV processes, facing each stria, smaller than the areolae (multiple arrowheads); c broken SV with a large copula (arrowhead), SVVC still attached to the SV, with no fimbriae (ellipse); d Detail of the SV processes (arrowhead), note that the SV is possibly bi-layered (framed arrowheads). Scale bars = 2 µm (a), 1 µm (b–c), 300 nm (d)

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Fig. 5
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Cocconeis vaiamanuensis sp. nov. SEM (Raivavae, Ruatara rock). a RV in external view. Whole valve with radiate striae, marginal hyaline area (white arrowhead), one row of marginal elongate poroids, denser than the striae (black arrowhead); b simple terminal raphe ending (arrowhead), absence of elongate poroids on apex (ellipse); c high cingulum (arrowhead); d anchor-like and raised fold around the simple terminal raphe ending (arrowhead); e proximal raphe endings small and bent on primary side (twin arrowheads) ; f RV striae with small areolae in quincunx ending in a pyramidal shape before the marginal hyaline area; g central area absent-missing. Scale bars = 2 µm (a, c), 1 µm (b, e–f), 700 nm (g), 600 nm (d)

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Fig. 6
figure 6

Cocconeis vaiamanuensis sp. nov. SEM (Raivavae, Ruatara rock). a RV in internal view. Whole valve with a small portion of the RVVC, apparently with no fimbriae (arrowhead), low helictoglossa; b proximal raphe endings curved in opposite sides; c unraised marginal hyaline area (arrowhead); d domed RV areolae, in quincunx (arrowhead); e crenulated periphery of the RV hymenes (arrowhead); f probable edge of the RVVC slightly undulated, with no fimbriae (framed arrowheads); g detail of apex without poroids (ellipse). Scale bars = 2 µm (a, c), 1 µm (b, g), 600 nm (f), 200 nm (d–e)

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Family: Cocconeidaceae Kützing, 1844

Genus: Cocconeis Ehrenberg, 1837

Description

Frustule solitary, valve oblong-elliptic to linear in the larger specimens (SEM, n = 60, length 7–15 μm, width 3–5 μm, L/W 2.2), with round apices. Never observed in girdle view during this survey.

Sternum valve (SV)

slightly concave (Fig. 3), with a narrow, flat and straight sternum. Small round areolae (ca. eight per stria; 4–5 in 1 μm). SV striae uniseriate (n = 32, 16–22, x̄ 19.6 ± 1.3 in 10 μm), parallel in mid-valve to radiate and slightly denser on apices. Striae composed of tiny areolae, more or less in zig-zag, internally closed by strongly convex hymenes without obvious slits or punctuations (Fig. 4b, c). Running all over the valve, a marginal embossed apical and hyaline area similar to a crista marginalis (Fig. 3, arrow). Mantle narrow with one row of small pores providing access to a process (Fig. 3f, arrows). This small process is internally closed by a hemispheric plug or hymen, slightly different and smaller in size than that of the SV areolae (Fig. 4d, arrowhead). One process regularly faces each stria (Fig. 4b, arrowheads). Strong virgae, externally embossed on their most marginal part (Fig. 3d, arrowheads). Relatively high cingulum composed of several open and large and thin cingulae devoid of ornamentation (Fig. 3, arrowhead, c, e). SVVC apparently with no fimbriae (Fig. 4c, arrowhead and ellipse). SV valve possibly bi-layered (Fig. 4d, framed arrowheads).

Raphe valve (RV)

strongly convex (Fig. 5c). Striae radiate, slightly denser on apices (n = 30, 20–24, x̄ 21.3 ± 1.2 in 10 μm), composed of small areolae, biseriate near the axial area, uniseriate on a short median section, and up to quadriseriate near the margin. On rare specimens (one observed, see supplementary Fig. 11), the RV striae are distinctly biseriate, with alternate areolae. RV areola hymenes internally convex with a crenulated border giving it a star-like shape (Fig. 6d, e, arrowheads). A marginal row of dense and apically elongate-oblong poroids (60–72, x̄ 65.2 ± 3.5 in 10 μm, Fig. 5, black arrowhead), separated from the rest of the valve by a large hyaline apical area (Fig. 5, white arrowhead), corresponding to an internal flat unraised rim (Fig. 6). Central area reduced to absent (no evident fascia, Fig. 5, or short hemi-fascia on some specimens). Raphe filiform, straight. Proximal raphe endings externally close to each other (slightly bent on the same side, Fig. 5e) and internally bent in opposite directions (Fig. 6b). Terminal raphe endings simple, close to the margin (Fig. 5b, arrowhead), apically surrounded by an anchor-like silica fold (Fig. 5d, arrowhead) with no connection to the interior of the valve. No RV areolae on apices (Fig. 5b, ellipse). RVVC taking place on the border of the RV, with no fimbria (Fig. 6, arrowhead), or a slightly undulated edge (Fig. 6f, framed arrowheads). Central area almost absent (Fig. 5). Helictoglossa straight and low (Fig. 6g).

Holotype:

Whole slide from the sample RAI 20, deposited at NHM (BM 81917 material). Holotype illustrated in Fig. 5.

Type locality:

Ruatara rock (Rocher de l’Homme), Raivavae (Austral Islands, South Pacific), RAI 20 (intertidal red macroalgal turf on small fissures of the rock). Geo localization: S 23° 51.274′; W 147° 39.595′; T°C 23°.7, salinity < 40‰. Sampled by C. Riaux-Gobin on 08 10 2018.

Ecology:

Relatively rare, marine intertidal taxon, living on rocky shore covered by turf. The new taxon was present in RAI 20 (see above) but also, in lower densities, on RAI 18 (same GPS position-geo localization, on red-dark short algae), and RAI 21 (same geo-localization, red-brown algae, longer than those in RAI 18). Taxonomy of the varied short macroalgae (turf) not available.

Etymology:

The epithet vaiamanuensis refers to the most famous motu from Raivavae, the motu Vaiamanu (in Tahitian “the place where there are birds”), also named “motu Piscine.”

Distribution:

Until now only found on Raivavae, as a possible local endemic [i.e., to the best of our knowledge, absent in Rapa (Austral Islands), as well as in the other visited Polynesian Archipelagos. Also absent from Mascarenes and Scattered Islands (Indian Ocean), and other tropical locations referenced, i.e., in Riaux-Gobin et al. (2011c, p. 7).

Remarks:

Relatively rare taxon (< 5% of the all benthic diatom assemblage in RAI 20; occasionally present only on three samples in the vicinity of Ruatara). Some dissimilarity concerning the ultrastructure of the valves with Cocconeis nugalas MH Hohn & Hellerman (Hohn and Hellerman 1966) [synonym C. hauniensis Witkowski (Witkowski 1993; Desianti et al. 2015; see also SEM in Riaux-Gobin and Romero 2003, pl. 41–43]. The SV sternum is elliptical and slightly concave in C. nugalas while narrow-linear and flat in the new taxon. Cocconeis nugalas has no crista marginalis. The SV virgae are more significantly embossed in the new taxon than in C. nugalas. Furthermore, on C. vaiamanensis the RV striae are biseriate on a part of their length and composed of tiny areolae, while bigger and uniseriate on C. nugalas (Riaux-Gobin and Romero 2003: pl. 43, Fig. 3). The RV of C. vaiamanuensis has a margin with almost four oblong poroids between each stria, seemingly more complex than in C. nugalas where there is only one marginal oblong poroid between each stria. The hyaline marginal area is narrower in C. nugalas. Furthermore, the valve shape is different between the two taxa: elliptical in C. nugalas and linear-elliptical to rod-like in the new taxon. The two taxa are thus significantly dissimilar.

Based on LM, similarities also exist with Cocconeis finmarchica Grunow in Cleve & Grunow (Cleve and Grunow 1880, p. 16, pl. 1, Fig. 1; illustrated here in Fig. 2q), also a small taxon, with a narrow and straight SV sternum and a SV hyaline marginal area (possibly a crista marginalis), and an RV hyaline margin. The RV of the latter has a slightly denser RV striation than the SV (24 in 10 μm, 20 in 10 μm in the SV) and radiate RV striae, such as in the new taxon. The only points that do not match the new taxon are the narrow and long fascia on the RV in C. finmarchica, and the shape of the frustule that is ellipsoid. No recent bibliography offers SEM for C. finmarchica.

For Cocconeis with SV processes, and concerning the taxonomic key presented in Riaux-Gobin et al. (2015), the addition of a supplementary group would be necessary to identify the new taxon: i.e., “Group 5 with SV striae composed of numerous areolae, with no partition, and with a narrow sternum void of areolae.” This key would clearly benefit from references to the structure of the RV when available. However, neither C. inaequalistriata Riaux-Gobin, OE Romero, Compère & Al-Handal (Riaux-Gobin et al. 2011c, p. 28–29, pl. 48, Figs. 1–6; Riaux-Gobin et al. 2015, Fig. 13), nor C. sp4 in Riaux-Gobin et al. (2015), Figs. 12, 30–32; also illustrated in Riaux-Gobin et al. 2011c,? Cocconeis sp. 1, p. 41, pl. 85, Figs. 1–4) match the new taxon.

The genera Psammothidium and Platessa also have simple terminal raphe endings, but none correspond to our marine taxon. A cladistic analysis, based on ultrastructural characters (see below and Fig. 7), allows us better to understand the degree of proximity of genera and taxa. A phylogenetic tree including a taxon pertaining to the Cocconeis peltoides group also allows for some remarks to be made (see below and Fig. 8).

Fig. 7
figure 7

Cladistic Analysis based on ultrastructure. OG = out group. See comments in the text

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Fig. 8
figure 8

Phylogenetic tree, showing the position of different groups of Cocconeis. See comments in the text

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Cladistic analysis based on ultrastructure (Tables 3, 4, Fig. 7)

It can be first pointed that several selected characters (Table 3) may appear as blurry, such as the valve shape (difficult to define accurately) or curvature of the frustule in cingular view (along the apical axis), a major-determinant character often neglected in descriptions. The morphology of the valvocopulae (with or without fimbriae), also an important character, is not often detailed.

Table 3 Taxa (34 species), character coding (16 ultrastructural characters), author and year, where can be found the SEM documents referring to each taxon). OG = out group
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Table 4 All strains used for the phylogenetic analysis, including the taxon name, strain ID, and assession number (rbc L) in genbank
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Planothidium juandenovense is the outgroup, with groups of areolae with a multiseriate arrangement on the SV mantle and terminal raphe fissures strongly bent.

Platessa bahlsii Potapova (Potapova 2012) is the only taxon in the analysis showing multiseriate striae, seemingly belonging to Planothidium but with simple terminal raphe endings. Platessa bahlsii appears as an independent taxon (monophyletic). It would be recommended to culture such a taxon and to do molecular analyses to evaluate its degree of affiliation (proximity) to Planothidium. Kulikovskiy et al. (2020) recently suggested that “P. bahlsii belongs to the group of species without sinus or cavum in the genus Planothidium”, and proposed the new combination Planothidium bahlsii (Potapova) Kulikovskiy, Glushchenko & Kociolek, in Kulikovskiy et al. (2020).

The cladistic analysis (Fig. 7) roughly has two poles: (1) Group I composed of taxa pertaining to Platessa, Cocconeis without SV processes and Achnanthidium and (2) Group II composed of Psammothidium, Cocconeis with SV processes and other atypic taxa upon which Rossithidium that is not accepted as different from Psammothidium by various authors (see Kulikovskiy et al. 2016; Jüttner et al. 2020).

Illustrating the difficulty of establishing limits between Achnanthes Bory, 1822, Psammothidium, Platessa and Cocconeis, we can cite the intricate taxonomic history of Cocconeis therezienii Le Cohu & R.Maillard ex Van de Vijver & Le Cohu (Van de Vijver and Le Cohu 2019). The interpretative analysis of a taxon by different authors may illustrate the difficulty of defining it correctly. For example, Platessa oblongella (Østrup) CE Wetzel, Lange-Bertalot & Ector (Wetzel et al. 2017) and its synonym Karayevia oblongella (Østrup) Aboal, in Aboal et al. (2003), do not appear at the same level in the cladistic analysis (Fig. 7), due to SEM observations that do not exactly match.

Molecular phylogenetic position of Cocconeis cf. sigillata SZCZCH1200 and some other Cocconeis (Figs. 8, 9)

Our rbcL-based phylogenetic trees recover monoraphid diatoms as polyphyletic and spreads them over two diatoms clades: Achnanthes + Bacillariaceae sister to Cocconeidaceae + other monoraphid and raphid diatoms (100% ML bootstrap, 0.5536 BI posterior probability), although the support of some branches within the two clades is very low.

Fig. 9
figure 9

Detail of the position of Cocconeis cf. sigillata MT015687 (SZCZCH1200) in the phylogenetic tree (Fig. 8). See comments on the text

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As mentioned, Achnanthes as representative of Achnanthaceae is placed within the large clade together within Bacillariaceae family as sister to Nitzschia Hassall, 1845, Psammodictyon DG Mann, in Round et al. 1990 and Tryblionella W Smith, 1853, however, with a low support (< 50% ML bootstrap, 0.5035 BI posterior probability). This position of Achnanthes spp. is stable on either single gene (Mann et al. 2020), three genes (Theriot et al. 2015; Ashworth et al. 2017) or in multigene (Theriot et al. 2010; Nakov et al. 2018a, b) trees.

Cocconeidaceae, represented in our phylogeny by several strains of Cocconeis Ehrenberg, are spread over a few groups and are seemingly polyphyletic. The major Cocconeis-bearing clade is recovered fairly isolated and is positioned at the base of the clade containing all monoraphids and raphids, excluding Bacillariaceae. Here, Cocconeis pediculus Ehrenberg, 1838, and C. placentula Ehrenberg, 1838, create a fairly distant and monophyletic group, which is in a sister relationship to Cocconeis sp. KC309551 (strain name MPA2013ECT3901, see Table 4) (81% ML bootstrap, 1 BI posterior probability), and together they are grading into a smaller group composed of Berkeleya hyalina (Round & ME Brooks) EJ Cox, 1975 (< 50% ML bootstrap) and two Planothidium and Cocconeis cf. cupulifera Riaux-Gobin, OE Romero, Compère & Al-Handal, 2011c, KT943680 (strain name SZCZCH662) (< 50% ML bootstrap). The relationship of the latter taxon to Planothidium spp. is moderate (65% ML bootstrap, 1 BI posterior probability).

Psammothidium, Rossithidium and some Achnanthidium form a paraphyletic group with low support at the nodes but usually moderate to high (> 60% ML bootstrap, > 0.68 BI posterior probability) in the particular clades. This clade is grading further into a monophyletic branch composed of Achnanthidium (10 species), and another clade of Pauliella taeniata (Grunow) Round & Basson, 1997, both with moderate to high support. Cocconeis cf. sigillata MT015687 (strain name SZCZCH1200) and C. cf. mascarenica Riaux-Gobin & Compère, 2008 KT943679 (strain name SZCZCH283) (73% ML bootstrap, 0.9999 BI posterior probability) are nested within this clade (though, with low bv), with unresolved trichotomy including Lemnicola hungarica (Grunow) Round & Basson, 1997, and Cocconeis sp. KT943614 (strain name SZCZP67) (here with also very low bv). Sister to the above monoraphid clades is Planothidium related to the clade of Cymbellales, however, with a low support (54% ML bootstrap, 0.9812 BI posterior probability).

Cocconeis stauroneiformis Okuno, 1957, AB430694 (strain name s0230) is positioned within Surirellales and hence fairly distant from the above monoraphids and the major Cocconeis clades. Although, the relationship is weakly supported (< 50% ML bootstrap, 0.7763 BI posterior probability).

Finally, Schizostauron Grunow, 1867, Astartiella Witkowski, Lange-Bertalot & Metzeltin in Moser et al. 1998, Madinithidium Desrosiers, Witkowski & Riaux-Gobin, in Desrosiers et al. 2014, Karayevia and Kolbesia Round & Bukhtiyarova ex Round, 1998, belong in a monophyletic clade together with Stauroneidaceae as well as Parlibellus EJ Cox, 1998 and Fistulifera Lange-Bertalot, 1997.

Discussion

It can be noted that among the morphological characters chosen to delineate groups of taxa in our ultrastructural cladistics analysis (Table 3, Fig. 7), the absence of SV processes (i.e., in Platessa bahlsii Potapova, 2012, P. itoupensis Tudesque, Le Cohu & CE Wetzel, 2016, P. kingstonii Potapova, 2012, Cocconeis alucitae Riaux-Gobin & Compère, 2008, C. neuquina Frenguelli, 1942, C. coronatoides Riaux-Gobin & OE Romero, in Riaux-Gobin et al. 2011b) does not tightly group the latter taxa. Thus, it proves that a more determinant character (than the absence of SV processes), or a combination of characters, better drives the analysis. In the same manner, the terminal raphe endings (simple or showing either more or less large and bent fissures) do not seem determinant to group or individualize taxa.

Following this cladistic analysis, Cocconeis peltoides and affiliated taxa (with SV processes), are close to Psammothidium levanderi (Hustedt) Bukhtiyarova & Round, 1996 and P. toroi S Blanco, Pla-Rabès, CE Wetzel & I Granados, 2017. In contrast, Cocconeis taxa without SV processes (such as C. alucitae, C. neuquina and C. coronatoides) are part of another clade, close to several Platessa and far from Achnanthidium.

Our phylogenetic tree (Fig. 8) suggests that the genus Cocconeis is not monophyletic. Surprisingly C. cf. cupulifera SZCZCH662 (see Riaux-Gobin et al. 2011c, p. 24) has no genetic affiliation to C. cf. sigillata SZCZCH1200. In contrast, they both have SV processes and pertain to the same morphological group. Also surprising is the place of C. cf. mascarenica SZCZCH283 (a taxon without SV processes, cf. Riaux-Gobin and Compère 2008) that locates as a sister of C. cf. sigillata (Fig. 8). Several previous investigations (Nakov 2014; Kulikovskiy et al. 2016, 2019; Thomas et al. 2016; Witkowski et al. 2016; Shi et al. 2018) also showed that C. placentula Ehrenberg, 1838 and C. pediculus Ehrenberg, 1838 group together and that C. stauroneiformis Okuno, 1957 pertains to another clade. Finally, and as previously stated by, i.e., Kulikovskiy et al. (2016), the Order Achnanthales arbitrarily groups numerous genera that are not genetically inter-connected, and is thus a polyphyletic group. In more general terms, in support of Thomas et al. (2016), “the monoraphid diatoms are not a natural group.” Shape convergence strongly influenced past taxonomy, which needs to be revisited in light of genetic approaches. Nevertheless, genetic and morphological approaches (Figs. 7, 10) may complement each other, since the cultivation, followed by sequencing of numerous taxa (i.e., small marine taxa, particularly monoraphid ones) has up to now been impossible (no entries in GenBank), yet essential to clarify their phylogenetic position. Therefore, as a first step, the cladistic analyses based on ultrastructure can help to provide preliminary keys.

Fig. 10
figure 10

Cocconeis cf. sigillata MT015687 (SZCZCH1200). a SV in external view, with biseriate striae, crista marginalis and SV processes on the mantle; b SV in internal view, with an elliptic SV sternum void of areoalae; c RV in external view; d RV in internal view with two shorter striae between each stria. Striae biseriate near the raphe. Note the Voigt discontinuities (d). Scale bars = 5 μm

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