﻿Craspedostaurosnazmii sp. nov., a new diatom species (Bacillariophyta) from the Turkish Coast of the Black Sea

﻿Abstract Craspedostauros E.J. Cox is a diatom genus comprising 17 taxa reported from various regions of the world. While many species of Craspedostauros are epibiontic, the taxa have variable ecological preferences. In this study we formally describe Craspedostaurosnazmiisp. nov., an epilithic species discovered along the Turkish Black Sea Coast, based on light and scanning electron microscopy. Craspedostaurosnazmiisp. nov. is characterized by valves that are lanceolate to narrowly lanceolate, slightly constricted near the apices with uniseriate, parallel throughout the whole valve, transapical striae and and the presence of an apical silica flap. The areolae are distributed over the valve face and the mantle. The differences and similarities between C.nazmiisp. nov. and established species of Craspedostauros are discussed. Based on shape and morphometrics, the most similar species is Craspedostauroscapensis, but it is easily distinguished from C.nazmiisp. nov. by its lack of an apical silica flap.


Introduction
The genus Craspedostauros E.J. Cox, 1999 was established to accommodate several marine species previously included in Stauroneis Ehrenberg (Lange-Bertalot and Genkal 1999; Cox 1999).As the latter genus comprised mostly freshwater species, a separation was proposed by different authors to accomodate the marine species with regards to their autecology and morphological differences.Species strictly conforming with Stauroneis Ehrenberg are characterized by naviculoid valves with two girdle appressed chloroplasts and possess a distinct and large central area called stauros (Round et al. 1990).The valves in Stauroneis are flat and the girdle is composed of a few plain copulae that is always rather narrow (Krammer and Lange-Bertalot 1986;Round et al. 1990;Cox 1999).
When establishing the genus Craspedostauros, Cox (1999) referred to cytological (shape and number of chloroplasts) and ultrastrutural characteristics (cribrate occlusions of areolae), also leading her to point out an affinity with Mastogloia Thwaites ex W. Smith (1856).With regards to these affinities, PhytoKeys 232: 77-88 (2023), DOI: 10.3897/phytokeys.232.106545 Elif Yılmaz et al.: Craspedostauros nazmii sp.nov from the Turkish Coasts of the Black Sea Craspedostauros has been included in Mastogloiaceae with a recommendation to also move Achnanthes Bory sensu stricto (Cox 1999;Cox and Williams 2006) there.The molecular phylogeny published by Ashworth et al. (2017) and inferred from a 3 genes dataset (18S, rbcL and psbC genes) tends to associate Craspedostauros to Achnanthes and Staurotropis, but clearly distinguishes them from Mastogloia spp., strongly questionning the monophyly of the Mastogloiales.This group of genera was later referred to as CAS genera, for Craspedostauros-Achnanthes-Staurotropis by Mann et al. (2021), whose phylogenetic works surprisingly tended to associate the CAS genera with Bacillariaceae.However, the lack of support at the nodes or strong morphological evidences led the authors to consider this result unlikely, probably resulting from an artefact of genes/species sampling and they emphasized the need for deeper phylogenomic investigations in order to elucidate the position of the CAS genera.
As a genus, Craspedostauros is relatively small, with 15 taxonomically accepted species listed on AlgaeBase, to which could be added two recently described taxa from Antarctica (Trentin et al. 2022).Thus, there remains a potential for the description of new species in unexplored habitats such as tropical coasts and biofilms on seaweeds in particular (Risjani et al. 2021;Witkowski unpublished observations).
The Turkish Republic is surrounded by four different seas, namely the Eastern Mediterranean Sea, the Aegean Sea, the Marmara Sea and the Black Sea.Craspedostauros decipiens was found in the Sea of Marmara (Witkowski et al. 2000;Akçaalan and Kaleli 2021) but this is so far the only species of Craspedostauros discovered in Turkish Black Sea waters.The Black Sea is a semi-closed sea located in southeastern Europe.It is considered an isolated sea since the Dardanelles and Bosphorus straits limit water exchange with the Mediterranean Sea.This semi-closure influences both sea water characteristics (typically salinity) as well as the dispersal potential of aquatic species inhabiting both sides of the straits (Nevrova et al. 2013).Studying the diatoms of the Black Sea may be of additional interest for science in that they may represent flora of an ancient marine basin isolated due to limited water exchange over a fairly long geological time period (Witkowski et al. 2010).
From the list above, it is obvious that it is mostly the northern part of the Black Sea whose diatom communities have been investigated and in contrast, the Southern Turkish Coasts of the Black Sea only received attention very recently (Baytut 2013).The latter authors investigated the discharge zone of the Kizilirmak River into the Black Sea and among the diatom species list many new records for Turkey were published.Similarly, Kaleli et al. (2017) studied Akliman city in the Sinop area and provided a species list also with new records for Turkish waters.
In this article, we contribute to the expanding list of novel taxa by describing Craspedostauros nazmii sp.nov., a new epilithic species from the Turkish Coasts of the Black Sea.The results are based on light and Scanning Electron Microscopy.This is the first and, for now, only species of Craspedostauros observed along the Turkish Black Sea coast.

Material and methods
The sample was collected in July 2017 from epilithic substrata in Kastamonu Doganyurt, Southern Black Sea (42°0'29.24"N,33°27'34.19"E)(Fig. 1).A sin- gle epilithic sample was collected using a toothbrush from the surfaces of submerged stones of this sampling station.Environmental parameters were measured using a Lange Hach HQ40d.No live observations of the samples were conducted as the sample was processed directly and boiled with H 2 O 2 and 10% HCl to remove organic matter and calcium carbonate respectively.After washing the diatoms with distilled water several times, permanent slides were mounted with Naphrax synthetic resin.Light Microscope (LM) observations were conducted on an OLYMPUS BX51 Light Microscope with OLYMPUS EP50 camera at Kütahya Dumlupınar University.Scanning Electron Microscope (SEM) observations were made using a FEI Versa 3D at İstanbul University with secondary electron and backscatter excitation, 10 kV and a working distance 10 mm.For this purpose, samples were placed on polycarbonate membrane filters with a 5 μm mesh.The membranes were left to dry and then attached to aluminum stubs with double-sided carbon tape, and sputter coated with ca.20 nm gold using a Turbo-Pumped Quorum Q 150OT ES coater.Isotype: Slide number TR_Kastamonu_Doganyurt_EPL_Tem2017 deposited in Kütahya Dumlupınar University (Turkey).
Description.LM (Fig. 2A-M) valves lanceolate to narrow lanceolate, slightly constricted in the middle and with rostrate to subcapitate apices, 29-42 µm in length, 4.5-5.5 µm in width (n = 50).Valves with a slight constriction in the middle, tapering towards narrowly rostrate to subcapitate apices.Axial area very narrow, but distinct, in the valve middle expanding into a central area in a form of stauros encompassing the whole valve width.Raphe branches in LM resolvable, slightly undulate, external proximal raphe endings distinct, tear-like shaped, external distal raphe endings strongly bent in same direction.Transapical striae well resolvable in LM, parallel in the middle, becoming slightly radiate and finally divergent close to apices, 20-21 in 10 µm (Figs 2A-M, 3A-D).
SEM (Fig. 3A-H).Valve external view (Fig. 3A-D), valve face flat composed of one to two rows of areolae.The valve face margin marked by a distinct, continuous apically oriented siliceous rib (transformed vimineae).The transition from the valve face to valve mantle gradual in the valve middle, becoming abrupt at the apices.Hyaline area becoming larger towards to the margins in the centre.Transapical striae uniseriate, composed of 1-3 areolae on the valve face and 4-6 on the mantle in central area, and decreasing to 4 towards the apical mantle (Fig. 3B-D).The striae of the valve face in the apical part composed of a solitary areola, and increasing towards the valve middle to 2 and finally 3 near the stauros.Areolae variable in size, larger near the raphe with more pores (up to 17) in the cribrate occlusions (Fig. 3B).Raphe branches slightly undulate with external proximal ends expanded, distant from each other.External apical raphe endings strongly hook-shaped.Prominent wing-like silica flaps partially covering the first row of areolae bordering the raphe sternum present near the apices at valve secondary side (Fig. 3C-D).Valve centre with hyaline area of  the stauros and symmetric with regular areolae.On one side three and on the other one to two rows of areolae at the beginning.Then, two rows of areolae on both sides and finally one row of areola towards the ends (Fig. 3A-D).
SEM (Fig. 3E-H).Valve surface internally flat, narrow pore-free longitudinal lines running from apex to apex abruptly marking the face-mantle junction (Fig. 3E).Raised stauros distinctly narrower than the fascia (Fig. 3F), broadening and decreasing in thickness close to the valve margins (Fig. 3E-F).Central internal raphe fissures terminate at slight knob of silica onto rectelevatum (Fig. 3F).A flatly ended cylindrical knob present at the central nodule.Apical raphe endings terminating within prominent helictoglossae within a relatively expanded pore-free area (Fig. 3G-H).Areolae externally occluded by cribra, appearing sunken, especially close to the raphe-sternum (Fig. 3F-H).
Etymology.This species is dedicated to Nazmi Yilmaz, father of the first author Elif Yilmaz in appreciation for his dedication to support and encourage her.
Distribution and ecology.The species was observed in Doğanyurt District, Kastamonu Province, Black Sea.The conductivity values at the sampling station were 18.69 mS cm -1 , DO values were 8.86 mg L -1 , TEMP values were 15.4 °C.

Discussion
The taxa belonging in Craspedostauros originate from various geographic regions of the world with Craspedostauros britannicus E.J.Cox 1999 known

Figure 1 .
Figure 1.Map of the sampling location A shows the Kastamonu Province (red rectangle) on the shores of the Black Sea.The red dot on B indicates the exact place where the sampling was conducted in Doğanyurt, north of Kastamonu.Figures obtained from Google Maps, Creative Commons CCO Licence, GNU Free Document Licence.

Figure 3 .
Figure 3. SEM micrographs of Craspedostauros nazmii sp.nov.A external view of entire valve B external view of central area C, D external view of valve apex showing wing-like silica flap E internal view of entire valve F internal view of central area showing raphe terminate G-H internal view of valve apex.Scale bars: 10 μm (A, E); 2 μm (B-D, F-H).

Table 1 .
Comparison of the main morphological and morphometric characters of Craspedostauros nazmii sp.nov.(n = 50) with morphologically similar taxa from the literature.