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Three new Licmophora species (Bacillariophyta: Fragilariophyceae) from Guam, two with an axial wave in the valve

Diatoms in genus Licmophora are found as epiphytes on marine shores and are members of fouling communities. They can be abundant on natural coral reef substrates such as filamentous algae and as fringes on Halimeda. Biodiversity of benthic tropical diatoms is generally poorly known. Several new species of Licmophora have been reported from Guam but many are still undescribed. Samples of seaweeds with evident diatom coatings were collected during scuba dives, examined live, preserved in formalin, and processed with nitric acid cleaning following standard practice. Cleaned samples were examined in LM and SEM. We describe two species unusual in having an axial wave in the valve, and a further long, straight species. L. repanda n. sp. and L. undulata n. sp. were of similar lengths (ca. 200 μm long) and both had axial waves but L. undulata was more slender with an inflated basal pole, had colonies on mucilage pads vs. long, single-stranded mucilage stalks. In addition, L. undulata had one apical rimoportula vs. two, elongated areolae along the midrib vs. uniform areolae, and the valvocopular advalvar striae ended halfway along, whereas in L. repanda they continued to the end. L. joymaciae n. sp. grew on long, single-stranded mucilage stalks with valves 300–400 μm long, with two apical rimoportulae, and basal pole not inflated. As in L. undulata its valvocopular advalvar striae ended halfway along the band. Stria densities were different at apex vs. base in L. joymaciae but not in the two wavy species. In all species, the 4th pleura had an apical cap and a narrow band with a single row of pores but it was larger in L. joymaciae. The other three girdle bands did not present any major differences among the three species but are documented for future comparisons with other species. Although the morphometric differences are small among the new species and between them and similar known species, the combinations of character states clearly shows that they are different.


Introduction
Diatoms of the genus Licmophora C.A.Agardh are common and sometimes abundant epiphytes on marine shores from the poles to the tropics. As members of "fouling" communities they have attracted the attention of applied scientists (Daniel et al. 1987;Woods and Fletcher 1991;Zargiel and Swain 2014;Ravizza and Hallegraeff 2015). On natural substrates diatoms including Licmophora can be abundant under some circumstances, e.g., on the filamentous algae cultivated by pomacentrid farmer fish (Lobban and Jordan 2010). In Guam, and probably other tropical locations, Licmophora spp. commonly form fringes around the edges of living Halimeda segments, along with genera such as Climacosphenia and Ardissonea (Lobban et al. 2011). Some species form extensive blooms, e.g., L. colosalis Belando et al. (2016) and L. labianatis Lobban et al. (2018).
Licmophora cells are heteropolar in being clavate or spathulate in valve view and attached at the base. They are also heterovalvar, with a basal rimoportula on one valve so that front/back and left/right can be recognized in addition to the epitheca and hypotheca (Lobban et al. 2018). Valves are generally straight, but there are a few curved species, including our recently described L. curvata Lobban et al. (2018).
Diatoms with wavy outlines, where the valve outline has a wave form (as opposed to species where the wavy margins are the result of inflations, such as Grammatophora undulata Ehrenberg) are rare. They include Toxarium undulatum Bailey ex Bailey (Mediophyceae) and Neosynedra tortosa (Grunow) D. Williams & F.E. Round (Fragilariophyceae); in the latter the valve margins undulate but the sternum is straight. One species of Licmophora, L. flucticulata Lobban et al. (2011), has a wavy outline along part of the valve, but because of the weak silicification it is not clear whether the sternum is straight or follows the curves of the valves. L. subundulata Mereschkowsky (1901Mereschkowsky ( -1902, has a completely different outline.
In published records of tropical floras, the diversity of Licmophora appears to vary wildly. For example, Hagelstein (1939) reported but did not illustrate 19 taxa from Puerto Rico and the U.S. Virgin Islands; Giffen (1980) likewise listed 9 taxa from Mahé; Navarro (1982) showed 1 species from Puerto Rico; Hein et al. (2008) 3 species from The Bahamas. One difficulty with these lists, especially those lacking illustrations, is that we cannot tell whether the species truly correspond to the European taxa. For instance, L. ehrenbergii, widely reported from tropical locations, appears to occur in Guam but did not match European specimens at the ultrastructural level (Lobban 2013vs Honeywill 1998. The difficulties with determining the identity of tropical species are that most of the known species of Licmophora were described from European shores and there are relatively few characters on valves, even when ultrastructure is included. We recently showed that there are many useful characters in the girdle bands (valvocopula and pleurae) (Lobban et al. 2018), but this information has yet to be collected for most (including all European) taxa. Few species have yet been sequenced, and most of those were not European samples. Meanwhile, we have proceeded to analyze Guam Licmophora species that have distinctive features in the valve. Here we report on two species that have an axial wave in the valve and on a further long, straight species.

Methods
Study samples were collected, processed, and observed by light microscopy (LM) fresh and following preservation, and in SEM whole mounts and acid-cleaned material following standard protocols used in the Lobban lab (Lobban 2015a, b). In brief, samples preserved in formalin were rinsed, boiled with nitric acid and rinsed to neutrality. Drops of the resultant suspension were dried onto cover slips for LM and cellulose nitrate filters for SEM. LM observations were made with a Nikon 80i microscope with differential interference contrast (Nikon Instruments, Redmond, WA, USA). SEM observations were made with a desktop Phenom G2 Pro (PhenomWorld US, Hillsboro, OR, USA).
Terminology follows Lobban et al. (2018) and standard works cited therein.

Etymology
Latin, repanda = having a slightly uneven and waved margin.

Morphology
Samples were available from several collection times from GabGab and several different locations in and near Apra Harbor. Characteristics of valves and girdle bands from different samples were alike. Frustules were attached by long, single-stranded stalks. Plastids were elongated discs (Fig. 1b).
Valves ( Fig. 1) were 190-215 μm long, 17 μm wide at apex gradually tapering from apex to base, basal pole not inflated (Fig. 1a, d-f, h, i). Multiscissura with 23-26 slits (Fig. 1h, i). Striae 25-30 in 10 μm, areolae 12-14 in 10 μm, fairly uniform ( Fig. 1c, j). Three rimoportulae, all ca. 0.8 μm across inside (Fig. 1i, k),  Fig. 1k), and changes in midrib width. f, g The 4th pleura showing apical cap and portion of band with single row of pores. Scale bars 5 μm the two apical ones at the end of the sternum on the edge of the valve face, oriented along one of the radiating striae; external opening hard to distinguish (Fig. 1j). Basal pole width increased from 4.8 μm at end of multiscissura to 5.2 μm 10 μm from base, and continued gradually increasing towards apex. One to two waves were usually present (Fig. 1a, e-g), but we observed two populations (GU44AL-2A and GU44AR-3) that were straight but proved to be otherwise indistinguishable from L. repanda.
Valvocopula (Fig. 2a-e) with a narrow septum (0.9-1.3 μm wide) (Fig. 2a), open ends tapering abruptly and symmetrically (Fig. 2e), closed end with broad ligule on abvalvar side (shown as unnamed species in Lobban et al. 2018, Fig. 2). Copular striae 36-37 in 10 μm, apparently rimate on both sides of the midrib all the way to the open ends, where rimae becomes shorter. Midrib straight, asymmetrical, transition gradual: close to abvalvar margin near the apex but starting to move toward advalvar margin within 10 μm, completed ca. one third along the band.
4th pleura (Figs. 1l and 2f-h) comprised a shallow apical cap and a narrow band with one or two rows of pores and no midrib (see also apical view of whole mount of this species, then unnamed, in Lobban et al. 2018, Fig. 2).
The 1st-3rd pleurae did not have features that were useful in distinguishing among the species described here and in Lobban et al. (2018). However, the taxonomically useful pleura characters have not yet been established for the genus, so they may be useful in comparison with other species. We thus include brief descriptions here and have put the three associated plates in Additional files 1, 2 and 3.
1st pleura (Additional file 1A-D). Band bluntly rounded to tapered at open ends and closed end narrow with two rows of pores around the basal pole. Striae rimate; midrib symmetrical except tending toward abvalvar at open tips, wider in the middle and lower portions (ca. 800 vs 400 nm), then narrowing as band narrows toward basal pole (closed end).
2nd pleura (Additional file 1E-H): Band with broad ligules on both sides at the apex the advalvar ligule more clearly defined and raised into a narrow ligule with an apparently wider margin, though in apical view of whole mount there appears to be a narrow ligule on both margins. Striae rimate except around the apex, abvalvar ends of striae uneven across the ligule. Midrib symmetrical except strongly deflected toward the advalvar side around apex (Fig. 1k). Midrib narrow around apex, broadest about half-way along the band.

Morphology
Cells attached individually or in small groups by mucilage pads with oval plastids (Fig. 4b). Valve length ranged from 150 to 200 μm and 10-15 μm wide at the apex, gradually tapered from apex to base with an inflated basal pole (Figs. 3 and 4a, c, g, f, j, k). Multiscissura with 20-24 slits (Fig. 4j, k). Striae 30-35 in 10 μm; areolae markedly long along the sternum (Fig. 4h), ca. 7x longer than wide (where wide is in apical direction). Areolae 10-12 in 10 μm. Apical rimoportula at end of sternum on the edge of the valve face, small, apically oriented. Basal rimoportula further from pole (2 striae) than in L. repanda (Fig. 4j). Basal pole width at apex of multiscissura ca. 4.2 μm, but 10 μm from base ca. 2.5, whereas in L. repanda, width of pole was slightly wider and got continually wider. In L. undulata, there was a ± parallel section until the first wave, then gradually wider. Valvocopula (Fig. 5a-e) with narrow septum (ca. 1.25 μm) (Fig. 5a), advalvar striae ended halfway along the band (Fig. 5c), leaving a wide margin and midrib on advalvar side, alongside the abvalvar row of slits. Midrib asymmetrical throughout, deflected toward the abvalvar side around the apex (Fig. 5b). The midrib did not get wider but became continuous with the abvalvar margin.

Etymology
Named for the senior author's mother, Joy Macatugal, who encouraged the author to pursue this undergraduate research opportunity.

Morphology
Colonies on long single-stranded mucilage stalks; frustules narrowly cuneate in both valve and girdle view. Plastids numerous small oval plates.
Valves (Fig. 6) 300-400 μm long, 17-20 μm wide at apex gradually tapering from apex to base (Fig. 6a, e, f ), basal pole not inflated (6d, j, k). Stria density 30-35 in 10 μm at the apex and 26-28 at the base, pattern of areolae uniform (Fig. 6c, h, i). Apical rimoportulae on edge of valve face beyond the sternum, oriented more or less in the apical plane along one of the radiating striae (Fig. 6h, i); Fig. 6j and the relative sizes of internal and external openings suggest the rimoportula is a funnel about 1.5 μm across inside. Basal rimoportula at end of sternum, on the right, also about 1.5 μm wide. Multiscissura with 26-29 slits (Fig. 6j, k).    White arrow shows midrib of 2nd pleura swung towards advalvar side and black arrow shows where abvalvar striae are lost on 4th pleura. h External view of apex with rimoportula opening (arrow), also showing portion of the 4th pleura (arrowheads). i Internal view of apex with rimoportula and uniform pattern of striae along septum. j Oblique internal apex showing shape of rimoportula. k, l External view of basal pole with rimoportula opening (arrow). m Internal view of basal pole with rimoportula. Scale bars: a, e, 50 μm. b-d, 10 μm, f, 100 μm, g-i, k-m, 5 μm, j, 2.5 μm Valvocopula (Fig. 7a-e) with a narrow, solid septum (2.75 μm wide) (Fig. 7a). Copular striae 41-42 in 10 μm. Midrib gradually moved from abvalavar to advalvar margin (Fig. 7a), eliminating the advalvar striae about halfway along the band (Fig. 7d, e).
4th Pleura (Figs. 6g and 7f-h): Band comprised a relatively large apical cap and a narrow band with initially two rows of rimae, the abaxial one soon ending (Fig. 6g).
1st Pleura (Fig. 6g Lobban et al. 2018Honeywill 1998 and others Belando et al. 2016Hustedt 1931Honeywill, 1998Lobban et al. 2011 a For species studied here, n = 10 unless specified otherwise b n/a = not applicable (2015) called for species-specific approaches toward mitigating the impact of fouling diatoms: species-conditions combinations that produce the most mucilage have the greatest impact on human society. But it is clear that one cannot get far with such work in tropical environments until there is much better knowledge of the diversity of the genus around the tropics.