Chaetoceros decipiens Cleve 1873, p. 11

Li, Yang, Boonprakob, Atchaneey, Gaonkar, Chetan C., Kooistra, Wiebe H. C. F., Lange, Carina B., Hernández-Becerril, David, Chen, Zuoyi, Moestrup, Øjvind & Lundholm, Nina, 2017, Diversity in the Globally Distributed Diatom Genus Chaetoceros (Bacillariophyceae): Three New Species from Warm-Temperate Waters, PLoS ONE (e 0168887) 12 (1), pp. 1-38 : 5-26

Chaetoceros decipiens Cleve 1873, p. 11 , Pl. I, fig 5a & 5b, emend. Li, Boonprakob, Moestrup & Lundholm

Figs 1– 3 , 20E and 20F and S 1 and S 2 Figs.

Synonym: Chaetoceros grunowii Schütt 1895

Lectotype designated here. A holotype was not chosen by Cleve. We have chosen slide MIC 5366 in P. T. Cleve’s collection, Stockholm, Sweden as the lectotype. The slide is labelled “Atlanten ytan 27/5-71 Lat 60˚25 Long 19˚50 ThM Fries”. Figs 1A, 1B and 2A illustrate the lectotype.

Examination of the type material revealed the morphology to be in agreement with Cleve’s description and with our strains identified as C. decipiens . The Cleve material from the North Atlantic and the Davis Strait looked similar. Most prominently some sibling setae were found to fuse for a longer distance; however, in others no fusion was seen. A very delicate striation of the setae was observed, in agreement with Cleve [ 7]. We did not at first observe the striation in our cultured material, but a closer examination showed a similar and extremely delicate striation.

Type locality: North Atlantic 60˚25’N 19˚50’W

Epitype designated here: Glutaraldehyde-fixed material of strain P10E5 isolated from the Norwegian Sea (67,9050N, 4.3238W). The material has been deposited at the Natural History Museum of Denmark, Copenhagen (C-A-92068). Fig 1C and 1D illustrate the epitype. A sequence of D1-D3 LSU rDNA represents the epitype (Genbank accession number KX065223) .

Emended description: Chains are usually straight, sometimes slightly arc-shaped curved ( Fig 1A, 1C, 1E and 1F ). In broad girdle view, the cells are quadrangular or rectangular ( Fig 1A, 1B and 1D–1G ) sometimes with the apical axis longer than the pervalvar axis ( Fig 1F ), sometimes the reverse ( Fig 1B, 1D, 1E and 1G ). Solitary cells also occur ( Fig 3A ). Several (3– 12) chloroplasts are present in each cell ( Fig 1E and 1F ). In valve view, valves are broadly elliptical to round-oval; the valve face is saddle-shaped ( Figs 2F and 3B ), the central region slightly raised and higher than the valve face margin. A silica rib along the valve face margin is broadly arcshaped ( Figs 2F and 3B ). On the valve face, the costae diverge while anastomosing from a central annulus, with solitary poroids scattered between the costae ( Fig 2H ). The intercalary

valve corners touch those of the adjacent cells ( Fig 1B and 1D–1F ). The apertures are variable, narrow slit-like oval to hexagonal ( Fig 1B and 1D–1G ).

Setae are stiff and extend from the corners of the cell ( Fig 1A–1F ). All setae of a chain are located more or less in the apical plane (Brunel group I) [ 34] ( Fig 1A–1D ), sometimes diverging very slightly from the apical plane ( Fig 1E ). Sibling setae cross over just outside the chain border and may fuse for a shorter or longer distance ( Figs 1A–1D , 3B and 3C ). The setae lack a basal part ( Figs 1B, 1D and 3C ). The extent of fusion varies even within a single chain and between the two sides of adjacent cells ( Fig 1D , arrowheads, Fig 3B ). The terminal setae diverge, forming an open U ( Figs 1A, 1C, 1F and 3A ). On the intercalary valve, a silica membrane of variable size is present at the margin of the apertures, forming a continuation of the marginal silica rib. The membranes of sibling cells may overlap to form a junction between the cells ( Fig 3B , arrow, Fig 3C ). Silica fringes are present on the mantle below the membranes ( Fig 3C , arrowhead) and are more distinct on the terminal valves, sometimes with long protuberances ( Fig 2F , arrowheads, Fig 3A ). Four to six rows of poroids and spines are arranged in longitudinal rows along the setae ( Fig 2C–2E ). The poroids are round-oval ( Fig 2C–2E ), 0.3 ±0.1 μm long in size and with a density of 19.0±6.7 poroids in 10 μm (n>50). Sometimes a striation is visible under LM ( Fig 2A ), sometimes not ( Fig 2B ), not reflecting poroid density, but more or less similar to the density of the spines ( Table 1 ). All setae have the same structure.

A single rimoportula without an external tube is situated centrally on the terminal valve ( Fig 2I , arrowhead). No processes were observed on the intercalary valves ( Fig 2H ). Sometimes, V-shaped non-silicified protrusions can be seen in LM centrally on the terminal valves ( Fig 1G , arrowhead). In LM, a constriction is visible at the border between the mantle and the girdle bands ( Fig 1B and 1D , arrows), and the mantle occupies approximately one third of the pervalvar axis. The mantle has narrow parallel rows of costae separated by single rows of poroids ( Fig 3D ). Silica warts are present on the basal ring of the mantle ( Fig 2G ).

The girdle bands have parallel costae separated by single rows of scattered poroids ( Fig 2J ). The apical axis of the valve is 7.8–64.3 μm long, the pervalvar axis 7.8–78.9 μm long, the length of the aperture in the pervalvar axis 3.3–15.1 μm (n>100). No resting spores were found.

Sexual reproduction was observed, and was thus homothallic (S1 and S2 Figs). Auxospores adhered to the girdle of the mother cell (S1 and S2 Figs, arrows). New daughter cells achieved a larger apical axis of the valves (S2 Fig).

Geographical distribution: Davis Strait, North Atlantic [ 7]; Disko Bay, Greenland (April, present study); Denmark Strait; Beaufort Sea; Norwegian Sea; Denmark (April, present study; most of the year with a maximum in spring [ 9]); Narragansett Bay of Rhode Island [ 10]; Gulf of Naples, Italy ([ 35], present study); Peter the Great Bay, Sea of Japan [ 13]; Japanese coast [ 36]; Pacific coast of Mexico [ 12]; southern Gulf of Mexico [ 15]; Daya Bay, south China (December, present study).

Chaetoceros elegans Li, Boonprakob, Moestrup & Lundholm sp. nov. Figs 4–7

Formal diagnosis: Straight chains or solitary cells. Four to ten chloroplasts typically present in each cell. Apical axis 11.7–39.7 μm. Pervalvar axis 8.9–42.2 μm. Aperture in pervalvar axis 4.4–14.5 μm. Cells quadrangular in girdle view. Saddle-shaped valve face. Central annulus, diverging costae and scattered poroids on valve face, continuing onto mantle. Silica rib on the valve face edge. A rimoportula present on the terminal valve. Furrow above the basal ring of mantle. Large and rounded, quadrangular-rectangular apertures. Setae in the apical plane. Basal part of setae extend in the pervalvar direction. Sibling setae cross over outside chain border without fusing. Terminal setae diverge in direction of chain. Silica ear-like structures present on base of setae. Four to six rows of poroids and spines on the four-six sided setae. Tear-shaped to elongate poroids on setae, ca. 0.5±0.2 μm in size and 17.8±5.4 poroids in 10 μm. Several bands, each band with parallel costae and scattered poroids. Resting spore with smooth surface. The primary valve extends into two elongated elevations with dichotomous branching processes. The secondary valve with one or two bulges. The angle of the outer slope of the elevation is acute. Length of elevation is 1–2 times longer than the branching processes.

Holotype: Glutaraldehyde-fixed material of strain YL7 deposited at the Natural History Museum of Denmark, Copenhagen (C-A-92069). Figs 4A–4D , 5D–5G and 6A–6C, H illustrate the holotype. A sequence of D1-D3 LSU rDNA represents the holotype (Genbank accession number KX065232).

Type locality: Dapeng Bay , Guangdong Province, P. R. China .

Etymology: referring to the characteristic very elegant overall look of the chains and the resting spores.

The chains are straight and stiff ( Fig 4A ). Cells are quadrangular in broad girdle view ( Fig 4A–4D ). Solitary cells also occur ( Fig 4B ). Typically four to ten chloroplasts are present within each cell ( Fig 4C ). The valves are broadly elliptical to round-oval ( Fig 5A ) with a saddle-shaped valve face, as the central region is slightly raised ( Fig 5B–5E ). The valve face edge is broadly arc shaped and marked by an elevated silica rib ( Fig 5B–5E ). On the valve face, costae diverge from a central annulus, with poroids scattered in between ( Fig 5F ). A constriction is visible at the border between the mantle and the girdle bands ( Fig 4D , arrows). The mantle occupies nearly one third of the pervalvar axis, and is ornamented with narrow parallel rows of costae interspersed by single rows of poroids ( Fig 5G ). A ring-shaped furrow is present above the basal ring of the mantle ( Fig 5B–5E and 5G arrowhead). Apertures are large and rounded quadrangular-rectangular ( Figs 4C , 5B and 5C ).

All setae of a chain are located in the apical plane (Brunel group I) ( Fig 4A ) [ 34]. The setae protrude from the elongated corners of the cell ( Figs 4C , 5B and 5C ). The basal parts of the setae extend initially in approximately the direction of the pervalvar axis, before curving and crossing over ( Fig 5B and 5C ). Sibling setae diverge at an acute angle to each other, and cross over just outside the chain border without fusing ( Figs 4C , 5B and 5C ). Intercalary setae near the ends of the chain are directed slightly more in the direction of the chain ends ( Fig 4A ). The two terminal setae diverge slightly, continuing more or less in the direction of the chain ( Fig 4A and 4B ). On the terminal valve, two silicified, ear-like structures project from the base of the setae, one on each side ( Fig 5D , arrows), forming a continuation of the narrow silica rib on the valve edge. On the intercalary valves, these ‘ears’ of sibling cells overlap and form a junction between the cells, in some cases fusing ( Figs 5B, 5C , 6A and 6B , arrows). A small gap is sometimes seen between the crossing bases of sibling setae and the overlapping, ear-like structures ( Fig 6A and 6B ). The setae are four to six-sided, with four to six longitudinal rows of poroids and spines arranged alternatingly on the setae ( Fig 6D–6G ). The seta poroids are tear shaped ( Fig 6D–6G ), 0.5±0.2 μm long with a density of 17.8±5.4 poroids in 10 μm (n>70) ( Table 1 ). The poroids are visible in LM ( Fig 6C ). Poroids are smaller, oval and less numerous near the base of the setae ( Fig 6A and 6B ). All setae have the same structure.

A single rimoportula with a short external tube is situated centrally on the terminal valve ( Fig 5A, 5D and 5E ), while processes are absent on the intercalary valves ( Fig 5B, 5C and 5F ). In LM, a V-shaped non-silicified protrusion is visible centrally on the terminal valve ( Fig 4D , arrowhead). Several open girdle bands are present, each band ornamented with parallel costae, which are separated by single rows of scattered poroids ( Fig 6H ). The apical axis is 11.7–39.7 μm long, the pervalvar axis 8.9–42.2 μm long, the pervalvar axis including basal parts 17.1–31.9 μm long, the length of the aperture in the pervalvar axis 4.4–14.5 μm (n>80).

The resting spores are located centrally in the mother cells, touching the bands and sometimes the valves of the mother cell ( Fig 7A and 7B ). The surface of the resting spore is mainly smooth ( Fig 7C and 7D ). The primary valve extends into two elongated elevations with dichotomous branching processes, and one or two bulges are present on the secondary valve face ( Fig 7A–7D ). The elevations are 32.5–48.0 μm long, the branching processes 5.5–14.2 μm long, and the apical axis 32.5–48.0 μm. The angle of the outer slope of the elevation is acute

( Fig 7C and 7D ). Length of elevation is 1–2 times longer than the branching processes

( Table 2 ). Sometimes, several silica bulges are located at the bases of the two processes ( Fig 7C and 7D ). Each process branches into a tree-like structure with the distal tips pointed and possessing one or several hooks ( Fig 7F ). A single ring of puncta is located near the margin of the secondary valve mantle ( Fig 7E ).

Geographical distribution: Japanese coast (as C. decipiens in [ 36]); Gulf of California (as C. lorenzianus in [ 12]); New Brunswick Canada (present study); Concepción, Chile (October, present study); Dapeng Bay, south China (August, present study); Mannai Island, Thailand (June, present study).

Chaetoceros laevisporus Li, Boonprakob, Moestrup & Lundholm sp. nov. Figs 8–10

Formal diagnosis: Straight chains. Several chloroplasts in each cell. Apical axis 22.4–

46.3 μm. Pervalvar axis 13.2–42.5 μm. Aperture in pervalvar axis 6.3–12.0 μm. Cells rectangular in girdle view. Saddle-shaped valve face. Costae diverge from a central annulus on the valve face, continuing on to the mantle. Silica rib on the valve edge. A rimoportula present on the terminal valve. A furrow located above the basal ring of the mantle. Oval-peanut shaped apertures. Setae in the apical plane. Valve corners on sibling valves touch each other. Sibling setae cross over just outside the chain border. Terminal setae diverge in the direction of the chain. Silica ear-like structures and fringes present on the base of setae. Four to six rows of poroids and spines on the setae. Round-oval setae poroids, 0.6±0.1 μm in size, with 13.8±1.9 poroids in 10 μm. Several bands with parallel costae and separated poroids. Smooth resting spores with two conical elevations on the primary valve and one or two on the secondary valve.

Holotype: Glutaraldehyde-fixed material of strain N7 deposited at the Natural History Museum of Denmark, Copenhagen (C-A-92070). Figs 8A, 8D–8F and 9A–9G illustrate the holotype. A sequence of D1-D3 LSU rDNA represents the holotype (Genbank accession number KX065240).

Type locality: Mannai Island, Rayong Province, Thailand.

Etymology: laevis (Lat.): smooth, the resting spores are smooth without extensions.

The chains are straight and stiff ( Fig 8A, 8B and 8D ). In broad girdle view, cells are rectangular, the apical axis usually longer than the pervalvar axis ( Fig 8A and 8B ). Several chloroplasts (more than ten) are present within each cell ( Fig 8A ). The valves are broadly elliptical to round-oval ( Fig 8F ). The valve face is saddle shaped, as the central region is slightly raised ( Fig 8D ). The valve face edge is broadly arc shaped and marked by an elevated silica rib ( Fig 8D ). On the valve face, costae diverge from a central annulus, without poroids between the costae ( Figs 8F and 9C ). In LM, a constriction is visible at the border between the mantle and the girdle bands, and the mantle occupies approximately one third of the pervalvar axis ( Fig 8A , arrows). The mantle is ornamented with narrow, parallel rows of costae ( Fig 9C ). A circular furrow is present above a basal ring of the mantle ( Fig 9C , arrowhead). Valve corners on intercalary valves are elevated and almost touch those of adjacent cells ( Fig 8A and 8D ). Apertures are oval-peanut shaped ( Fig 8A, 8B and 8D ).

Setae of a chain are situated more or less in the apical plane (Brunel group I) ( Fig 8B ), sometimes diverging very slightly from the apical plane ( Fig 8A ). The intercalary setae are straight or slightly curved, those near the ends curving more towards the ends ( Fig 8B ). The setae vary greatly in length ( Fig 8B ). They protrude from the corners of the cell, and sibling setae cross over just outside the chain border, without any fusion ( Fig 8A and 8D ). An ear-like structure is present at the seta base of intercalary valves near the marginal border of the aperture ( Fig 9A , arrows). The ‘ears’ sometimes form a junction between sibling cells ( Fig 9B , arrow). The two terminal setae diverge slightly, continuing more or less in the direction of the chain ( Fig 8A and 8B ). The setae are four to six-sided, and four to six longitudinal rows of poroids and spines are arranged alternatingly on the setae ( Fig 9G ). The seta poroids are round-oval ( Fig 9G ), 0.6 ±0.1 μm μm long, with 13.8±1.9 poroids in 10 μm (n>25), visible in LM ( Fig 8C )( Table 1 ). Poroids near the seta bases are smaller and more scattered ( Fig 9B ). All setae have the same structure.

A single rimoportula, which lacks an external tube, is situated centrally on the terminal valve ( Fig 8E , arrowhead, Fig 8F , lower valve). In LM, a V-shaped non-silicified protrusion is visible centrally on the terminal valves ( Fig 8A , arrowhead). Processes are absent on the intercalary valves ( Fig 8F , upper valve). Siliceous fringes are present near the terminal seta base ( Fig 9C , arrows). Several open girdle bands are present ( Fig 9D ), each with parallel costae separated by two, occasionally three, rows of scattered pores, in addition to larger poroids ( Fig 9E and 9F ). The apical axis is 27.7–34.2 μm long, the pervalvar axis 13.2–42.5 μm long, and the length of the aperture in the pervalvar axis measures 6.3–12.0 μm (n>80).

The resting spores are located centrally in the mother cells, touching both valves and bands of the mother cell ( Fig 10A ). The spore surface is smooth with two conical elevations on the primary valve and one or two on the secondary valve ( Fig 10A and 10B ).

Geographical distribution: Daya Bay, south China (December, present study); Mannai Island, Thailand (December, present study), Gulf of Panama (as C. cf. lorenzianus in [ 35]).

Chaetoceros mannaii Boonprakob, Li, Moestrup & Lundholm sp. nov. Figs 11 and 12

Formal diagnosis: Short straight chains or solitary cells. Several chloroplasts in each cell. Apical axis 5.7–12.9 μm. Pervalvar axis 8.4–29.6 μm. Aperture in pervalvar axis 4.6–5.6 μm. Cells rectangular in girdle view. Saddle-shaped valve face. Heavily silicified frustule. Robust diverging costae on the valve face, continuing onto the mantle. Silica rib on the valve face edge. A rimoportula, with long external process on the terminal valve. The mantle occupies one fourth of the pervalvar axis. A furrow is present above the basal ring of the mantle. Hexagonal apertures. Setae in or slightly diverging from the apical plane. Short basal part present, sibling setae cross over outside chain border. Terminal setae diverge in the direction of the chain. Silica ear-like structures present on the base of setae. Four to six rows of poroids and spines on the setae. Large oval setae poroids, 0.7±0.2 μm in size, 12.3±1.6 poroids in 10 μm. Several bands with parallel costae and scattered pores.

Holotype: Glutaraldehyde-fixed material of strain N1 deposited at Natural History Museum of Denmark, Copenhagen (C-A-92071). Figs 11 and 12 illustrate the holotype. A sequence of D1-D3 LSU rDNA represents the holotype (Genbank accession number KX065246).

Type locality: Mannai Island, Rayong Province, Thailand.

Etymology: from Mannai Island, Thailand.

Short straight chains are typical ( Fig 11A ), but solitary cells also occur. Several chloroplasts (4–10) are present within each cell ( Fig 11A ). In broad girdle view, cells are rectangular ( Fig 11A ). In valve view, valves are broadly elliptical to round-oval ( Fig 11B ). The valve face is saddle shaped, as the central region of the valve face is slightly raised ( Fig 11C and 11D ). The valve face edge is broadly arc shaped and marked by an elevated silica rib ( Fig 11C–11E ). Robust costae diverge from the centre of the valve face, without poroids between the costae ( Figs 11F and 12C ), and continue onto the mantle as robust parallel longitudinal ribs ( Fig 12D ). In LM, a constriction is visible at the border between the mantle and the girdle bands ( Fig 11A , arrows), and the mantle occupies ca. one fourth of the pervalvar axis. The basal ring of the mantle is heavily silicified, with a distinct furrow above the ring ( Figs 11F and 12D , arrowheads). The apertures are hexagonal ( Fig 11A, 11C and 11D ).

Setae of a chain seem to be located more or less in the apical plane (Brunel group I), or sometimes slightly diverging from the apical plane ( Fig 11B ). The intercalary setae are straight or slightly curved ( Fig 11B ). The setae protrude from the elevated corners of the cell ( Fig 11C ). Sibling setae cross over just outside the chain border, with short basal parts present ( Fig 11C and 11D ). Terminal setae diverge in an acute to 90 degrees angle ( Fig 11A, 11E and 11F ). Silicified ear-shaped structures are located at the base of the setae on both the intercalary and terminal valves ( Fig 11D and 11E , arrowheads). The ‘ears’ form a continuation of the narrow silica rib on the valve face edge ( Fig 11E ). In the intercalary valves, the ears of sibling setae do not appear to overlap ( Fig 11C and 11D ). Setae are four-six sided with four to six longitudinal rows of poroids and spines arranged alternatingly on the setae ( Fig 12B ). The seta poroids are oval ( Fig 12B ), 0.7±0.2 μm long and with 12.3±1.6 poroids in 10 μm (n = 20), readily visible in LM ( Figs 11A and 12A )( Table 1 ). Those near the seta base are slightly smaller and more scattered ( Fig 12B ). All setae have the same structure.

A single rimoportula with a long external tube is situated centrally on the terminal valve ( Figs 11E, 11F and 12C ). Processes are absent on the intercalary valves ( Fig 11C and 11D ). Several open girdle bands are present ( Fig 12E ), each with parallel costae separated by one row of scattered pores ( Fig 12F ). The apical axis is 6.7–12.9 μm long, the pervalvar axis 8.4–29.6 μm long, the length of the aperture in the pervalvar axis 4.6–5.6 μm (n = 20). No resting spores were found.

Geographical distribution: Peter the Great Bay, Sea of Japan (as C. lorenzianus in [ 13]; Gulf of California (as C. lorenzianus in [ 12]; Sinaloa, Mexico (present study); near Mannai Island, Thailand (present study).

Chaetoceros mitra (Bailey) Cleve 1896 p.4 , Pl I, fig 6 emend. Li, Boonprakob, Moestrup & Lundholm Figs 13–15 and 20A–20C

Basionym: Dicladia mitra Bailey 1856

Synonym: D. groenlandica Cleve 1873

Lectotype designated here: fig 2, plate II in Cleve [ 18] (shown as Fig 20C ). A holotype does not exist and a lectotype has therefore been selected.

Epitype designated here: Fixed material of strain P10A1, from TromsØ, Norway. Material has been deposited at the Natural History Museum of Denmark, Copenhagen (C-A-92072). Figs 13–15 illustrate the epitype. Sequences of D1-D3 LSU rDNA (Genbank accession number KX065247) and SSU rDNA ( KX611427 ) represent the epitype .

Type locality: Sea of Kamtschatka

Chains are straight ( Fig 13A ), or slightly curved (not shown). Several chloroplasts are present in each cell (often 6–14). In broad girdle view, cells are usually rectangular, the pervalvar axis often longer than the apical axis ( Fig 13A and 13B ). In valve view, the valves are broadly elliptical to round-oval ( Figs 13C , 14G and 14H ). The valve face is saddle-shaped, as the central region of the valve face is slightly raised ( Figs 13C and 14G ). The valve face edges are broadly arc shaped and marked by an elevated silica rib ( Figs 13C and 14G ). On the valve surface, costae diverge from a central annulus ( Fig 14H ), without distinct poroids between the costae. A constriction is located at the border between the mantle and the girdle bands ( Fig 13B , arrows). The mantle occupies one third to one fifth of the pervalvar axis, but sometimes less–as little as one tenth during resting spore formation ( Fig 15A and 15C ). The mantle is ornamented with narrow parallel rows of costae ( Fig 14G ). A furrow is situated above the basal ring of the mantle ( Fig 14F and 14G , arrows). Apertures are narrow oval to hexagonal, sometimes slightly indented in the middle ( Fig 13A and 13B ).

The setae of a chain are diverge from the apical plane (Brunel group II) ( Fig 13A ). Setae are soft and more or less curved and protrude from the corners of the cell ( Fig 13A and 13B ). The terminal setae have almost the same orientation as the intercalary setae ( Fig 13A–13C ) or they are slightly V-shaped in broad girdle view. Sibling setae cross over at the chain border, with no basal parts ( Figs 13A, 13B and 14F ). Silicified wing-like structures are present near the seta base and form a bridge between sibling cells ( Fig 14F , arrowhead). They also form a continuation of the silica rib along the valve face edge on the intercalary valves. On the terminal valve they are replaced by fringes ( Fig 14G , curved arrow). The setae are four-six sided with four to six rows of poroids and spines arranged alternatingly on the setae ( Fig 14B–14E ). Poroids of the setae are round-oval ( Fig 14B–14E ), 0.2±0.1 μm in size, 39.8±7.4 poroids in 10 μm (n>20), the density varying within a single seta. The poroids are barely visible in LM ( Fig 14A )

( Table 1 ). Poroids near the seta bases are slightly smaller and more scattered ( Fig 14F ).

A single slit-like rimoportula without any external tube is situated slightly excentrically on the terminal valve ( Fig 14G , arrowhead). Processes on intercalary valves are absent ( Fig 14H ). Several open bands are present, each with parallel costae ( Fig 14I and 14J ). The apical axis is 16.5–23.8 μm, the pervalvar axis 28.1–48.2 μm, the length of the aperture in the pervalvar axis 2.9–10.0 μm (n>20).

Resting spores are most often situated close to one valve of the mother cell ( Fig 15A and 15C ), sometimes in the middle of the cell ( Fig 15B ). The surface of the resting spore is mainly smooth ( Fig 15E–15G ). The primary valve extends into two elongated elevations with dichotomous branching processes distally ( Fig 15B–15G ). One or two bulges are present on the secondary valve face ( Fig 15E and 15G ). The elevations are 21.8–36.2 μm long, the branching processes 5.7–11.4 μm long, the apical axis 14.6–21.4 μm ( Table 2 ). The outer slope of the elevation is almost straight ( Fig 15F and 15G ). Length of elevation is 2–5 times longer than the branching processes ( Table 2 ). A single circular row of small silica warts is visible along the secondary valve edge ( Fig 15E , arrowheads). The mantle touches the bands of the mother cell ( Fig 15A–15C ). A ring of puncta is present at the margin of secondary valve mantle ( Fig 15G , arrowheads).

Geographical distribution: Greenland (April, present study); Sea of Kamtschatka [ 17]; Cape Wankarema, east coast of Greenland, Baffin Bay [ 18]; Narragansett Bay of Rhode Island [ 10], Gulf of St. Lawrence; Canada (as C. lorenzianus in [ 11]).

C. lorenzianus Grunow 1863 p. 157 , Pl 14, fig 13 Figs 16 and 20D

Lectotype designated here: A slide in a capsule (Acqu 1901/3674) with a coverslip of mica found in Grunow’s accession book under number 501 (Grunow 501). The capsule is glued onto a small paper sheet, and next to the capsule is a sketch of C. lorenzianus made by Grunow similar to fig 13 in Grunow [ 8] (here illustrated in Fig 20D ). Fig 16A and 16B illustrate the lectotype. A holotype was not selected by Grunow.

Type locality: Adriatic Sea

Original description: Rectangular or quadrangular cells in girdle view, setae bend out, setae long, delicate with punctuation. Apical axis 20–43 μm.

Examination of the lectotype material: only a few frustules were found on the slide. (The mica was observed using a 40X objective without oil to avoid destroying it): The aperture was quadrangular-hexagonal ( Fig 16A ). The setae protruded from the elevated corners of the cell and were located more or less in the apical plane. Sibling setae crossed over just outside the chain border, with some fusion of the basal parts of the setae and with short basal parts present ( Fig 16A and 16B ). Seta poroids were large and visible in LM as punctuations ( Fig 16B ), with a density of 7.2±1.7 poroids in 10 μm.

Figures

Fig 1. LM of Chaetoceros decipiens. A, C, E, F: Chain views of lectotype material MIC5366 (A) and strains P10E5 (C), P14B3B (E), D12 (F). B and D: Detail of chains showing constrictions (arrows) between the mantle and the girdle bands,and the fused extensions of sibling setae, which are short, longer or even absent (arrowheads); lectotype material MIC5366 (B) and strain P10E5 (D). G: Chain view of strain D10, note the V-shaped protrusion located centrally on the terminal valve (arrowhead). A and C: scale bars, 50 μm. B, D, E, F: scale bars, 20 μm. G: scale bar, 10 μm. doi:10.1371/journal.pone.0168887.g001

Fig 2. Chaetoceros decipiens. LM (A and B), TEM (C, D, H-J) and SEM (E-G). A-E: Seta structure of lectotype MIC5366 (A), strains P14B3B (B) and D10 (C–E), showing the 4–6 sided seta with poroids and small spines. F: Terminal valve with fringes (arrowheads); strain D10. G: Silica warts on the basal ring of the mantle; strain D10. H: Annulus, costae and poroid pattern on intercalary valve; strain P10E5. I: Terminal valve showing rimoportula without external process (arrowhead); strain D10. J: Girdle bands; strain D10. A and B scale bars, 10 μm. C–J scale bars, 2 μm. doi:10.1371/journal.pone.0168887.g002

Fig 3. Chaetoceros decipiens. Strain D10, SEM (A–C) and TEM (D). A: Solitary cell with silica fringes.B: Intercalary cells with overlapping silica membrane (arrow). C: Detail of fused seta bases, silica membrane and fringes on the mantle (arrowhead). D: Rows of poroids on the mantle. A and B scale bars, 10 μm. C and D scale bars, 2 μm. doi:10.1371/journal.pone.0168887.g003

Fig 4. LM of Chaetoceros elegans sp. nov. Strain YL7. A: Chain view displaying seta divergence in the apical plane. B: Solitary cell. C: Chain view demonstrating large apertures and chloroplasts. D: End of chain showing constrictions (arrows) between the mantle and the girdle, and V-shaped protrusion (arrowhead) located centrally on terminal valve. A and B scale bars, 50 μm. C and D scale bars, 20 μm. doi:10.1371/journal.pone.0168887.g004

Fig 5. Chaetoceros elegans sp. nov. SEM (A–E) and TEM (F and G). A: Terminal valve view with external process of rimoportula and poroids on valve face; strain MC1048. B and C: Intercalary cells demonstrating large apertures and valve faces; strain M1 (B), strain MC1048 (C). D and E: Terminal valves with central processes (arrowheads) and silica ear-like structures (arrows in D) in strain YL7. F: Annulus, costae and poroid pattern near intercalary valve centre; strain YL7. G: Parallel rows of poroids on the mantle; arrowhead indicates ring-shaped constriction; strain YL7. A–E scale bars, 5 μm. F and G scale bars, 2 μm. doi:10.1371/journal.pone.0168887.g005

Fig 6. Chaetoceros elegans sp. nov. LM (C), TEM (A, B, H) and SEM (D–G). A and B: Overlapping ear-like structures (arrows) and small gap between the crossing bases of sibling setae in strain YL7. C: Terminal seta with partly visible poroids in LM; strain YL7. D and E: Seta structure showing elongated poroids (D, strain Ch12A1) and tear-shaped poroids (E, strain M1) and F and G: Detail of setae poroids; strain Ch12A1 (F) and strains MC785 (G). H: Girdle band; strain YL7. All scale bars are 2μm, except 10 μm in C. doi:10.1371/journal.pone.0168887.g006

Fig 7. Resting spores of Chaetoceros elegans sp. nov. LM (A and B) and SEM (C–F). A and B: Resting spores within mother cells in a chain; strain MC785. C and D: Released resting spores, showing two elongated elevations with dichotomous branching processes distally on the primary valve face and one (C) or two bulges (Fg D) on the secondary valve face; strain Ch12A1. E: Internal view of secondary valve with a ring of marginal punctae; strain Ch12A1. F: Hooks on the distal tips; strain MC785. A and B scale bars, 20 μm. C–E scale bars, 5 μm. F scale bar, 1 μm. doi:10.1371/journal.pone.0168887.g007

Fig 8. Chaetoceros laevisporus sp. nov. LM (A–C), SEM (D and E) and TEM (F). Fig A: Part of a chain showing constrictions (arrows) between mantle and girdle, and V- shaped protrusion (arrowhead) located centrally on the terminal valve; strain N7. B: Dividing chain showing seta divergence; strain DY1. C: Structure of terminal seta; strain DY1. D: Chain in girdle view showing cells and apertures; strain N7. E: Terminal valve with rimoportula without external process (arrowhead); strain N7. F: Valve views of intercalary (upper) and terminal valves (below); strain N7. A, C, D scale bars,20 μm. B scale bar, 100 μm. E and F scale bars, 5 μm. doi:10.1371/journal.pone.0168887.g008

Fig 9. TEM of Chaetoceros laevisporus sp. nov. Strain N7. A, B: Non-overlapping (arrows in A) and overlapping ears (arrow in B) between sibling cells. C: Terminal valve with silica fringe (arrows) and constriction (arrowhead). D and E: Open girdle bands.F: Detail of girdle band with pores. G: Seta with round-oval poroids. A, B scale bars, 2 μm. C–E, G scale bars, 5 μm. F scale bar, 1 μm. doi:10.1371/journal.pone.0168887.g009

Fig 10. Resting spores of Chaetoceros laevisporus sp. nov. LM (A) and SEM (B). Resting spores within mother cells of a chain (A) and single, released resting spore (B); strain DY1. A scale bar, 20 μm. B scale bar, 5 μm. doi:10.1371/journal.pone.0168887.g010

Fig 11. Chaetoceros mannaii sp. nov. LM (A and B), SEM (C and E) and TEM (D and F); strain N1. A: Straight chain showing seta divergence and constrictions (arrows) between the mantle and the girdle. B: Oval valve face. C and D: Intercalary cells, with ear-like structures (arrowheads in D) at the bases of setae in heavily silicified frustule. E and F: Terminal valves, with ear-like structures at the seta bases (arrowheads in E) and distinct constriction above the ring (arrowheads in F). A scale bar, 20 μm. B–F scale bars, 5 μm. doi:10.1371/journal.pone.0168887.g011

Fig 12. Chaetoceros mannaii sp. nov. LM (A), SEM (B) and TEM (C–F); strain N1. A and B: Setae with oval poroids. C: Terminal valve showing valve structure and rimoportula with external projection. D: Poroids and costae on the mantle. E and F: Girdle bands with poroids. A scale bar, 10 μm. B scale bar, 5 μm. C–F scale bars, 2 μm. doi:10.1371/journal.pone.0168887.g012

Fig 13. LM of Chaetoceros mitra. Strain P10A1. A: Straight chain showing seta divergence and apertures.B: Detail of chain showing constrictions (arrows) between mantle and girdle. C: Valve structure of terminal valve. A and B scale bars, 20 μm. C scale bar, 10 μm. doi:10.1371/journal.pone.0168887.g013

Fig 14. Chaetoceros mitra. LM (A), SEM (B, C, F, G) and TEM (D, E, H–J); strain P10A1. A–E: Setae showing round-oval poroids and spines, using different microscopy techniques. F: Intercalary valves showing wing-like structures (arrowhead), and furrows above the basal ring of mantle (arrows). G: Terminal valve showing rimoportula without external tube (arrowhead), furrow above the basal ring of mantle (arrows) and fringe (curved arrow). H: Intercalary valve face. I and J: Girdle bands. A scale bar, 10 μm. B, C, F–H scale bars, 5 μm. D, E, I, J scale bars, 2 μm. doi:10.1371/journal.pone.0168887.g014

Fig 15. Chaetoceros mitra resting spores. LM (A–C), TEM (D) and SEM (E–G); strain P10A1. A: Early stage of resting spore formation. B and C: Mature resting spores within mother cells. D: Two elongated processes with dichotomous branches distally. E–G: Resting spores in different views,showing a row of silica warts along the secondary valve edge (arrowheads in E) and a ring of puncta at the secondary valve mantle (arrowheads in G). A–C scale bars, 10 μm. D–G scale bars, 5 μm. doi:10.1371/journal.pone.0168887.g015

Fig 16. Lectotype material of C. lorenzianus from the Grunow Collection. A: Intercalary valves with oval-hexagonal aperture and some fusion of the basal parts of the setae. B: Intercalary seta poroids visible under the LM. Scale bars are 10 μm. doi:10.1371/journal.pone.0168887.g016

Fig 20. The original drawings of species of the C. lorenzianus complex. A: Resting spore of C. mitra by Bailey [17]. B and C: C. mitra by Cleve [18]. Vegetative chain, intercalary valve,terminal seta and apical view of seta positions (C), and chain with resting spores (B, lectotype).D: Vegetative chain of C. lorenzianus by Grunow [8]. E and F: Chaetoceros decipiens by Cleve [7]. G and H: Resting spores of C. lorenzianus by Okamura [39]. doi:10.1371/journal.pone.0168887.g020