Haslea berepwari Mouget, Gastineau & Jauffrais 2025, sp. nov.

Haslea berepwari Mouget, Gastineau & Jauffrais sp. nov. Type material. Holotype: The strain P 05 was acid cleaned and mounted on a glass slide and is deposited in the herbarium “ Paris Cryptogamie ” (PC) at The French National Museum of Natural History under accession number PC 0643624 (LM slide) and PC 0643625 (SEM slide). The cell representative of the type is presented in Fig. 2. Isotypes: SEM and LM slides with acid cleaned valves of strain P 05 are kept at the Ifremer culture collection in New Caledonia under the accession number P 05. Type locality. Boulouparis, New Caledonia. Haslea berepwari was isolated from shrimp earthen ponds (coordinates: 21°55'36.9"S, 166°05'00.9"E, Fig. 1) by Thierry Jauffrais in August 2020 in Boulouparis during a co-culture experiment of Penaeus stylirostris and Holothuria scabra. Etymology. The species designation is derived from the term “ Boulouparis ”, which is the one of the main cities on the west coast of New Caledonia. The name “ Berepwari ” is the translation of Boulouparis in xârâcùù, one of the main Melanesian languages spoken in New Caledonia. Description. LM Living cells solitary, motile and lanceolate, equipped with two parietal, narrow band-like chloroplasts appressed to the girdle of the cell (Fig. 2). Valves narrow and lanceolate with acute apices. The maximum and minimum length of the monoclonal culture of H. berepwari was 101.0 μm and 95.4 μm, respectively (average 98.0 ± 1.5 μm, n = 30), while the maximum and minimum width was 15.0 μm and 9.7 μm (average 12.2 ± 1.1 μm, n = 30). On clean frustules, raphe straight with non-distinct central endings. Cell wall exceedingly delicate, with longitudinal and transapical striations not discernible under LM. In general, LM provides minimal visibility into the specifics of the valve characteristics and is not sufficient to distinguish between this species and H. pseudostrearia. SEM In external valve view, the exterior is covered with long, continuous, and apical-oriented siliceous stripes (top layer), proximal raphe endings straight and slightly widened, slightly deflected dorsally, apical raphe endings ventrally hook shaped (Fig. 3 A, D). The interior is composed of a grate-like layer of small areolae, separated by short bars arranged crosswise. Transverse bars of this layer are almost equal in the transapical and longitudinal bars. The areolae are occluded externally by hymens and remnants of this membrane are visible in Fig. 3 D. The central area lacks a lateral extension (Fig. 3 A, D). Internally, the raphe is slightly elevated and straight, with well-developed helictoglossae at the poles (Fig. 3 E). Internal openings of the raphe fissures directed towards one side of the raphe sternum, except at the center and near the tips. Thin bar near the central ending of the raphe on one side of the valve only (Fig. 3 C). A supplementary ridge runs alongside the raphe sternum across most of the valve. Internally, square-shaped areolae organized in orderly rows (Fig. 3 B, C, E). Externally, the valve seems covered with longitudinal bands, separated by slits running parallel to the raphe and converging into a single peripheral slit near the tips (Fig. 3 A, F). The striation displays a transapical pattern of 37–38 striae per 10 µm intersected by a longitudinal pattern of 36 striae per 10 µm. Differential diagnosis. A comparative analysis of morphological features between H. berepwari, Haslea nusantara (Mouget, Gastineau and Syakti) and H. pseudostrearia is detailed in Table 1. Haslea berepwari sp. nov. shares strong similarities with H. pseudostrearia but is distinguished from it by the density of striae, both transapical and longitudinal. Genomics and phylogeny. The nuclear rRNA gene cluster: For reasons unknown, we failed to assemble the complete cluster of nuclear rRNA, even after adjusting the k-mer parameter for assembly. However, we successfully retrieved the complete 18 S gene and submitted it to GenBank (PP 725422). This sequence completely validated the results obtained previously from Sanger sequencing. The sequence was aligned using Clustal Omega (Sievers et al. 2011) with references ascribed to H. pseudostrearia (AY 485524 and KY 320350) and identity was respectively 95.12 % and 95.30 %, while these two references were 99.81 % identical with each other. Mitochondrial genome: The mitochondrial genome of H. berepwari was retrieved from the contigs file with redundant endings. After trimming and circularization (Fig. 4), its length is 36,572 bp (GenBank: PP 728232). The mitogenome encodes for 34 proteins, considering that nad 11 is split into two distinct subunits. As it was noticed with other species of Haslea spp., nad 6 and nad 2 are merged into a single open reading frame (ORF), for a total size of 753 amino acids (Gastineau et al. 2021 b; Dąbek et al. 2022). The mitogenome also encodes for three ORFs. The first one, orf 162, corresponds to the conserved ORF generally found mttB and rps 11 (Pogoda et al. 2019; Dąbek et al. 2022). We note that our annotation software (Gagnon 2004) ascribed it to rpl 10, a function suggested for this ORF in Pleurosigma sp. (QYJ 09263) (Wang et al. 2022). However, in the absence of more evidence of the function of this gene, we will keep labelling it as orf 162, nothing also that the size of the putative protein encoded is identical among all the species of Haslea spp. for whom a mitogenome is available. The two other ORF, namely orf 171 and orf 235, are interspersed between the two subunits of nad 11 and cox 3 and are similar to ORFs found in the same position among other species of Haslea spp. (Gastineau et al. 2021 b; Dąbek et al. 2022). InterProScan queries returned no results for orf 235. For orf 171, four transmembrane domains, three cytoplasmic regions and two non-cytoplasmic domains were found. The mitogenome also encodes 22 tRNA and two ribosomal rRNA. Plastid genome: The plastid genome is 131,897 bp long (GenBank: PP 728231) and exhibits the usual quadripartite structure (Fig. 5). The LSC is 65,599 bp long and contains 74 protein-coding genes and 17 tRNA. The SSC is 48,934 bp long and contains 52 protein-coding genes, a single non-conserved ORF and seven tRNA. The inverted repeats are 8,682 bp long and contains two protein-coding genes, a non-conserved ORF (orf 118), three rRNA genes and three tRNA. The noticeable differences when compared to H. pseudostrearia are the position of cplC (between psbA and ycf 35) and the absence of overlap between ycf 45 and the IRB. Multigene phylogeny: The 123 - genes ML phylogeny led to a highly supported tree in which all nodes display maximum support (Fig. 6). For the genus Haslea, the tree distinguishes between a highly supported clade of marennine-like producing species and a second clade that contains H. berepwari sp. nov. and H. pseudostrearia. It is noteworthy that the genetic distance between both species is rather important when compared to the distance between ‘ blue’ species. Other taxa registered as Haslea on GenBank are nested within Navicula spp., but their belonging to the genus Haslea has been invalidated in Li et al. (2017) and thus should be instead regarded as Navicula spp. It is to note that Seminavis robusta D. B. Danielidis & D. G. Mann 2002 appears inside the Navicula clade, a position already observed in the 3 - genes ML phylogeny recently published in Yılmaz et al. (2024 a). The three-genes ML phylogeny (Fig. 7) also associated H. berepwari to a clade formed by two strains ascribed to H. pseudostrearia with high support. Sister to this clade is Haslea arculata Lobban & Ashworth, 2020, a species found in the Island of Guam and which is characterized by the curved shape of its frustule (Lobban et al. 2020). This large clade is sister to the sigmoid species Haslea nipkowii (Meister) M. Poulin & G. Massé 2004 (Poulin et al. 2004) and Haslea feriarum M. A. Tiffany & F. A. S. Sterrenburg 2015, a species with dorsoventral valve shape (Sterrenburg et al. 2015; Li et al. 2017). The tree strictly separates ‘ blue’ and ‘ non-blue’ taxa.

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