Morphology and molecular phylogeny of Pleurosigma pacificum sp. nov. (Pleurosigmataceae), a new tropical pelagic species from the Western Pacific Ocean

A new species of pelagic diatom, Pleurosigma pacificum sp. nov. , is described from the tropical Western Pacific Ocean. It has the typical features of Pleurosigma , including a slightly sigmoid raphe, intersected transverse and oblique striae, and loculate areolae with external opening slits and internal poroids. Morphologically, P. pacificum belongs to a species group of Pleurosigma with lanceolate valves, including P. atlanticum Heiden & Kolbe, P. nubecula W. Smith, P. indicum Simonsen, and P. simonsenii Hasle. However, P. pacificum differs by its smaller lanceolate valve and smaller intersection angle as well as elliptical areolae without a silica bar. The SSU rDNA and rbc L sequence data place P. pacificum in a basal position relative to other species of Pleurosigma . Our molecular phylogenetic analyses did not support the monophyly of lanceolate and slightly sigmoid species. Thus, the sigmoidality of valve outline cannot be considered as a criterion to define the species group.

Morphology and molecular phylogeny of Pleurosigma pacificum sp. nov. (Pleurosigmataceae), a new tropical pelagic species from the Western Pacific Ocean Introduction Smith (1852) established the genus Pleurosigma W. Smith for some sigmoid naviculoid diatoms. Peragallo (1891) clarified the infrageneric delimitation by combining the orientation of the striae and their angle of the intersection. Species with transverse and oblique striae were separated from those with transverse and longitudinal striae as well as species with centrally interrupted striae. Subsequently, Cleve (1894) transferred all those species with transverse and longitudinal striae into the genus Gyrosigma and retained species with transverse and oblique striae in the genus Pleurosigma. Hendey (1964) gave a clearer delimitation of Pleurosigma and provided chloroplast characters. Round et al. (1990) and Reid (2012) used ultrastructural features for their description of Pleurosigma. Currently, Pleurosigma is characterized by having two or four ribbonlike plastids, (slightly) sigmoid valves, transverse and two obliquely intersecting striae and loculate areolae with external opeing slits and internal poroids (Round et al. 1990;Reid and Williams 2003;Sterrenburg et al. 2005;Reid 2012). To date, this genus contains more than 700 named taxa (Kociolek et al. 2021).
Pleurosigma is a group of diatoms that is widely disturbed in brackish to marine environments (Round et al. 1990;Sterrenburg et al. 2003). It is predominantly found as a large population on sediments (Reid 2012) with a sigmoid valve or raphe, but some Pleurosigma species have lanceolate or nearly straight valves and are commonly found in planktonic samples. Metabarcoding data have revealed that Pleurosigma species are highly diverse in pelagic ocean (Malviya et al. 2016). However, only five species are known to be pelagic, namely P. antarcticum Grunow, P. atlanticum Heiden & Kolbe, P. indicum Simonsen, P. simonsenii Hasle and P. directum Grunow (Cleve andMöller 1877-1882;Heiden 1928;Simonsen 1974;Sar et al. 2012).
In this paper, we describe a new species of Pleurosigma isolated from the tropical Western Pacific Ocean by using light microscopy (LM) and scanning electron microscopy (SEM). Its phylogenetic position is determined by DNA sequence.

Sampling, cultivation and morphological observation
Phytoplankton samples were collected from upper 200 m water column by using a phytoplankton net (64 µm mesh), on the Western Pacific Ocean (7°0.26'N, 141°59.63'E). Single cells of diatoms were isolated using capillary pipettes and cultivated in F/2 medium. Cultures were maintained at 24-26 °C under a light intensity of 120-150 µmol photon/m 2 /s, with a light/dark cycle of 12:12 h. Five milliliters of vegetative cells were fixed with 2.5% glutaraldehyde and then cleaned with hydrogen peroxide (Trobajo and Mann 2019). For LM observation, cleaned samples were mounted on glass slides with Mountmedia (Wako Pure Chemical Industries, Ltd., Osaka, Japan). A Zeiss Imager Z2 microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) with differential interference contrast (DIC) was used for LM observation. The measurement methods of the raphe angle and the intersection angle of the oblique striae followed Sterrenburg (1991). For SEM observations, specimens were placed on coverslips, air-dried and coated with osmium. A Hitachi S-4800 (Hitachi, Ltd., Tokyo, Japan) was used for SEM observation.

DNA extraction and sequencing
The DNA was extracted and sequenced according to the method described in Li et al. (2022). Algal cell pellets were obtained by centrifuged 10 mL diatom cultures at 5,000 g for 5 min. Total DNA was extracted by using the Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). The small-subunit ribosomal DNA (SSU rDNA), large-subunit ribosomal DNA (LSU rDNA), chloroplast-encoded genes rbcL and psbC were amplified by polymerase chain reaction (PCR). Forward and reverse strands were amplified using the follow primes (Table 1). The PCR cycles of the four markers follow Alverson et al. (2007). The PCR products were purified using TIANgel Midi Purification Kit (Tiangen Biotech Co., China) and sequenced by Tsingke Biotechnology Co.,Ltd. (Beijing, China).

Molecular phylogenetic analyses
To examine the phylogenetic position of P. pacificum, a two-gene dataset (SSU rDNA-rbcL) including 30 recognized species and 12 unnamed strains, was used to construct the phylogenetic trees (Suppl. material 1). Due to the lack of LSU rDNA and psbC data of most Pleurosigma species in GenBank, the two genes were not used for the phylogenetic analysis. Since a previous molecular phylogenetic study indicates that Pleurosigmataceae is closely related to Haslea and Navicula (Li et al. 2017), we selected all the available sequence of Pleurosigmataceae and Haslea species in GenBank for analysis. For Navicula species, we selected sequences of species with voucher slides or reliable morphological data.
The SSU rDNA and rbcL sequences were aligned using MAFFT v.7.313 (Katoh and Standley 2013) with normal mode and Q-INS-I strategy which considered the secondary structure of RNA, respectively. The trimAl was used to trim the alignment with parameter automated1 (Capella-Gutiérrez et al. 2009). The final concatenated alignment included 2,224 positions, of which 1,535 columns were SSU rDNA and 689 were rbcL. PartitionFinder 2 was used to select best-fit models for ML and BI analysis , according to the Bayesian information criterion (BIC). The rbcL gene was partitioned by codon position. IQ-TREE v.1.6.8 (Nguyen et al. 2015), Mrbayes v.3.2.7 (Huelsenbeck and Ronquist 2001) and TNT v.1.6 (Goloboff and Morales 2023) were used to perform maximum likelihood (ML), Bayesian inference (BI) and maximum parsimony (MP) analysis, respectively. The ML analysis with 1,000 bootstrap was executed with the default settings. The BI program was run for 10 7 generations with samples every 1,000 generations and the first 25% of trees were discarded as burn-in. Convergence was judged based on the average standard deviation of split frequencies (all less than 0.01) and the ESS values (more than 3,000) were analyzed in the R package RWTY (Warren et al. 2017). The consensus topology and posterior probabilities of all branches were derived from the remaining trees using a majority-rule consensus approach. In the MP analysis, we used a traditional search with TBR branch swapping on 1,000 replicates and holding 10 trees per replication. The resulting 31 most parsimonious trees (MPTs) were used to calculate a strict consensus tree; Standard bootstrap and Jacknife (with 35 removal probability) analyses were performed using a traditional search and 1,000 replicates, with outputs saved as frequency differences. FigTree v.1.4.4 and Adobe Illustrator were used to view and edit trees.
Holotype. MBMCAS286904, an example is illustrated in Fig. 1A, E, F. This slide was deposited in the Marine Biological Museum, Chinese Academy of Sciences, Qingdao, China.
Isotype. MBMCAS286905, an example is illustrated in Fig. 1C. This slide was deposited in the Marine Biological Museum, Chinese Academy of Sciences, Qingdao, China.
Type locality. 7°0.26'N, 141°59.63'E, the upper 200 m water column in the tropical Western Pacific Ocean.
Etymology. Named after the Pacific Ocean where the species was discovered.

Distribution and ecology.
Pleurosigma pacificum is a planktonic species known only from the type locality. The water temperature was 28.5 °C and salinity about 33.4 during sampling.

Molecular phylogenetic analyses
The BLASTn search showed that the SSU rDNA sequence of P. pacificum shares 97.65% identity with an uncultured marine eukaryote (KC771201). The rbcL gene sequence of P. pacificum shares 92.54% identity with P. intermedium (NC_066077). The ML, BI and MP phylogenetic tree based on the concatenated SSU rDNA and rbcL gene showed that P. pacificum belongs to the clade of the genus Pleurosigma with high support (IQ-TREE ultrafast bootstrap value = 99, Mrbayes posterior probability = 1.00, MP standard boostrap value = 77 and MP jacknife value = 85). The P. pacificum is basal to all other species of Pleurosigma, and branched earlier than the two slightly sigmoid species, P. planctonicum and P. intermedium (Fig. 2, Suppl. material 2).

Discussion
The valves of Pleurosigma pacificum sp. nov. possess intersected transverse and oblique striae, opposite curved raphe distal endings, two internal central bars, and loculate areolae with an internal poroids and an external slit opening. These are considered to be the main characters of the genus Pleurosigma (Hendey 1964;Reid 2002;Sterrenburg et al. 2005). Molecular data place P. pacificum in a basal position relative to other species of Pleurosigma. The morphological data place P. pacificum in a group of species, which includes P. nubecula W. Smith, P. indicum Simonsen, P. simonsenii Hasle, and P. atlanticum Heiden & Kolbe, with lanceolate valves and a straight raphe (Table 2). Among them, P. pacificum can be easily distinguished from P. simonsenii by its much smaller valves (45.0-51.5 µm vs. 300-600 µm long). P. pacificum differs from P. indicum as its internal poroids of areolae are not bisected by a central bar (Fig. 1G in this study vs. figs 41 and 42 in Sar et al. 2012), and from P. atlanticum and P. nubecula by the smaller stria angle (32-35° in P. pacificum, 60° in P. atlanticum and 60-61° in P. nubecula).
The definition of the genus Pleurosigma has undergone constant debate and modifications (Cleve 1894;Hendey 1964;Round et al. 1990;Reid 2012). Pleurosigma is characterized by its (slightly) sigmoid valve and raphe, two or four ribbon-like chloroplasts, areolae opening to outside by elongate slits and inside by a poroid, areolae arranged in decussate rows, and central internal raphe ending in a central nodule. Reid (2012) indiates that the equal thickening of the raphe sternum on both sides of raphe is a synapomorphic feature of Pleurosigma and further revised the generic definition based on morphological phylogenetic analyses. Although Reid (2012) did not include any slightly sigmoid Pleurosgima species, recent studies and the present work showed that all these lanceolate and slightly sigmoid Pleurosigma species share this synapomorphic feature as well as the other features mentioned above (Sar et al. 2012;Sterrenburg et al. 2015). However, our molecular phylogenetic analyses did not support the monophyly of these lanceolate and slightly sigmoid species (Fig. 2, Suppl. material 2). Therefore, the sigmoidality of valve outline cannot be considered as a criterion to define the species group.