New and interesting Luticola species (Bacillariophyta) from the mangroves of Nosy Be Island, NW Madagascar

Nosy Be Island is the largest (ca. 320 km²) coastal island off Madagascar, located off its NW coast. Along with a number of smaller islands, including Nosy Fanihy, Nosy Sakatia, Nosy Faly, Nosy Ambariobe, Nosy Tanikely, Nosy Komba, Nosy Mamoko and Nosy Tonga, it forms an archipelago, the islands of which originated as volcanoes (Melluso & Morra 2000; Collins & Windley 2002), probably during the Tertiary (Besairie 1968–69–71).

The diatoms of Madagascar are still rather poorly studied. The first studies, containing analyses of materials collected in 1904–2000, were published in 2002. Those surveys concerned only freshwater diatoms and at that time the authors identified 177 new freshwater diatom species (Metzeltin & Lange-Bertalot 2002). The knowledge about marine benthic diatoms from both Madagascar and Nosy Be Island is much more scant. However, there are publications on diatoms from areas close to the Madagascar region, including the analysis of 13 samples collected from Tanzania (Midwest Africa) near the city of Dar-es-Salaam and of one sample from Malindi in Kenia (Foged 1975), as well as a comprehensive study on marine diatoms of the Mascarenhas Archipelago (Mascarene Islands), concerning mainly La Réunion and Rodrigues Islands, where diatoms have been explored in various freshwater environments for many years (Coste & Ricard 1984; Klee et al. 2000), marine diatoms having been addressed since 2005 within the framework of the COSADIM (Coral Sand Diatoms off Mascarenes) project (Riaux-Gobin & Compére 2008; Riaux-Gobin et al. 2010a,b; 2011a,b).

The genus Luticola D.G. Mann, established in 1990 (Round et al. 1990), groups widespread species, from both fresh and brackish water. Levkov et al. (2013) revised the genus and described 91 Luticola species as new to science. In total, the monograph of Levkov et al. (2013) contains descriptions of about 200 species. Since then, new species have been described and new combinations in this genus have been developed (e.g. Zidarova et al. 2014; Glushchenko & Kulikovskiy 2015; Kohler et al. 2015; Bak et al. 2017; Chattová et al. 2017; Glushchenko et al. 2017; Straube et al. 2017). Taking into account the Luticola species listed in AlgaeBase (Guiry 2017) and those recently proposed as new to science, it is estimated that the genus currently contains about 220 species. Ecological, taxonomic, molecular and biogeographical studies of the genus are in progress (e.g. Kociolek et al. 2017; Wu & Bergey 2017).

Luticola species have rarely been recorded in our study area. A recent study on marine diatom assemblages of Nosy Be Island (Kryk 2016) revealed the presence of only two Luticola species, which are described herein. Previous studies from freshwater habitats of the Madagascar region (Metzeltin & Lange-Bertalot 2002) also identified only two Luticola species: L. mutica (Kützing) D.G.Mann in Round, R.M.Crawford & D.G.Mann (1990: 670) and L. muticoides (Hustedt) D.G.Mann in Round, R.M.Crawford & D.G.Mann (1990: 671) from Ivato. In addition, Metzeltin & Lange-Bertalot (2002) reported the third, unidentified Luticola species from Lac Mantasoa. However, more Luticola species have been recorded in the West Indian Ocean in the vicinity of Madagascar. A summary of the Luticola species reported from the region is presented in Table 1. The relatively small number of Luticola species in the region is likely due to the paucity of studies and suggests the need for further taxonomic revisions and research on Luticola species from Madagascar as well as from the entire Indian Ocean.

The present report describes LM and SEM observations on two Luticola species from a single sample collected from mangrove roots on Nosy Be Island. The sample contained numerous other taxa, both aerophytes, e.g. Humidophila (Lange-Bertalot & Werum) R.L.Lowe et al. (2014) and those tolerant of a high osmotic pressure, e.g. Tryblionella debilis Arnott

ex O’Meara (1873: 310). Ecology of the two Luticola new species can be inferred from measurements of environmental parameters taken during sampling.

The analyzed material was collected on 30 June 2014 as an epiphytic sample from mangrove roots at a site designated as NB4a, located near the mouth of a small river on the southern coast of Nosy Be Island (GPS coordinates: 13°24’0.06”S, 48°17’8.58”E; Fig. 1). The authors obtained a permission for sampling from the Ministry of Higher Education and Scientific Research, Antananarivo, Madagascar.

Figure 1

In the laboratory, the sample was treated with 10% hydrochloric acid (HCl) to remove calcium carbonate and, following thorough washing, boiled in 37% hydrogen peroxide (H₂O₂) to eliminate organic matter. After washing four times with distilled water, the final suspension was pipetted onto coverslips, left for evaporation, and mounted onto glass slides using Naphrax® diatom mountant. The slides were examined under a Zeiss Axio Scope A1 light microscope. Measurements and photographic documentation were performed with the Canon EOS 500D and Canon EOS Utility software. A total of 400 valves were identified to the level of species or variety (or numbered in the case of new species) and counted (Battarbee 1986; Bodén 1991) to assess the relative abundance of all species (including those new to science). Biodiversity indices (i.e. species richness, the Shannon H’ index and the evenness index) were calculated (Shannon 1948; Tuomisto 2012). For scanning electron microscope (SEM) observations, the cleaned material was pipetted onto 25 mm diameter Whatman® Nuclepore 2 µm mesh polycarbonate membrane filters attached to aluminum stubs and sputtered with 20 nm of gold using a Turbo-Pumped Sputter Coater Quorum Q 150OT ES. Diatoms were examined under a Hitachi SU 8010 SEM at the Podkarpackie Innovative Research Center of the Environment (PIRCE), University of Rzeszów.

During sampling, relevant environmental parameters (water pH, temperature, conductivity, salinity, redox potential and dissolved oxygen content) were recorded using the Multiparameter HANNA HI98194 device.

The new species are compared with similar taxa from around the world described in the relevant literature (Metzeltin & Lange-Bertalot 1998, 2002; Moser et al. 1998; Metzeltin et al. 2005; Levkov et al. 2013; Zidarova et al. 2014; Glushchenko & Kulikovskiy 2015; Kohler et al. 2015; Chattová et al. 2017; Glushchenko et al. 2017; Bak et al. 2017; Kociolek et al. 2017; Straube et al. 2017; Wu & Bergey 2017). The adopted terminology follows Round et al. (1990) and Levkov et al. (2013).

Luticola nosybeana and L. madagascarensis are similar in the valve outline, but differ in width (up to 7.5 μm in L. madagascarensis and up to 10.5 μm in L. nosybeana). Under LM, they can be distinguished by the shape of the central area (bow-tie shaped in L. madagascarensis and deltoid in L. nosybeana) and the isolated pores (while the isolated pore is robust and well visible in L. madagascarensis, it is not easily discernible in L. nosybeana). Under SEM, the two species are easily distinguishable due to different shapes and positions of the isolated pores (the external opening in L. madagascarensis slit-like, located in a surface depression, positioned halfway between the center and the valve margin; a round opening located close to the valve margin in L. nosybeana).

The valve outlines and the shape of the central area in specimens of L. nosybeana are remarkably similar to those in L. belawanensis Levkov & Metzeltin in Levkov, Metzeltin & A.Pavlov (2013: 74), also described from an island in the Indian Ocean (Sumatra). The two species differ primarily in the stria density, which is higher in L. nosybeana (20–24 vs. 18–20 in 10 μm), and the lack of continuation of proximal raphe endings to irregular, shallow grooves, butterfly-like (T-shaped) or insect-antennae-like (L-shaped) in L. belawanensis. Luticola kochiae Metzeltin in Levkov, Metzeltin & A.Pavlov (2013: 143) and L. similis Levkov, Metzeltin & A.Pavlov (2013: 219) differ from both our new species in the valve outline (more elliptic than lanceolate in L. nosybeana and L. madagascarensis) and, in the case of L. kochiae, also in the shape of the central area, larger and wider in L. kochiae. The shape of the proximal raphe ends in L. kochiae is typically hooked, whereas in L. nosybeana they are irregular or butterfly-shaped. The valves of L. nosybeana are smaller (length 9–27 μm, width 6.0–10.5 μm) than those of L. kochiae (14.5–33.0 and 8.0–14.5 µm). The shape of the central area in L. similis is different, more rectangular vs. deltoid, and the valves are more lanceolate, compared to those of L. nosybeana (elliptic). Moreover, L. kochiae and L. similis are known only from their type localities in Columbia and New Caledonia, respectively. In terms of the valve outline, L. nosybeana is also similar to a taxon shown in Levkov et al. (2013: 499) as “Luticola sp. aff. permuticoides Metzeltin & Lange-Bertalot” from Brazil, but differs in the shape of the central area (more rectangular in Levkov’s taxon) and in the proximal raphe ends (typically hooked and distant from each other in Luticola sp. aff. permuticoides).

The valve shape and striae arrangement in L. nosybeana are similar to those in many other Luticola species, e.g. L. hlubikovae Levkov, Metzeltin & A.Pavlov (2013: 130); smaller valves are in L. saprophila Levkov, Metzeltin & A.Pavlov (2013: 212), L. wetzelii Levkov, Metzeltin & A.Pavlov (2013: 257) and L. isabelae

Metzeltin & Levkov in Levkov, Metzeltin & A.Pavlov (2013: 140); however, these can be distinguished from L. nosybeana under both LM and SEM. Differences between these other Luticola taxa and L. nosybeana involve mostly shapes of the central area (which can be asymmetrical, deltoid or rectangular) and the external, round opening of the isolated pore located close to the valve margin in L. nosybeana, which is indiscernible under LM, but is robust and well visible in all similar taxa listed above. None of the similar taxa shows L- or T-shaped central raphe endings like those in L. nosybeana.

The valve outlines in the specimens of L. madagascarensis examined in this study are similar to those in L. similis described from New Caledonia. Both species have similar dimensions and densities of striae. The two species differ in their proximal raphe ends, which are short and expanded into small central pores in L. similis, while continue with irregular, shallow, elongated, L-shaped grooves in L. madagascarensis; the latter species is also characterized by a clearly visible elongated isolated pore. The proximal raphe fissures are bent in the opposite direction relative to the distal endings in L. madagascarensis. The two species differ also in their ecological preferences: L. similis was found in freshwater, a highly eutrophic and polluted environment (a tributary of the Hienghène River), while L. madagascarensis occurred in mangrove-associated water with salinity of 15 PSU. Luticola frequentissima Levkov, Metzeltin & A.Pavlov (2013: 112) shows similar proximal raphe endings, but they are shorter, more variable in shape, widened at the end, and pointing to the same side as distal endings (Noga et al. 2017). The distal fissures terminate on the valve face (not on the valve mantle as in L. madagascarensis), and the isolated pore of L. frequentissima is rounded and located near the valve margin, as opposed to the slit-like isolated pore located halfway between the valve center and valve margin in L. madagascarensis.

With regard to the valve outline, the shape of the valve apices and striation, L. madagascarensis resembles most closely several other Luticola species, such as L. tomesii Moser, Lange-Bertalot & Metzeltin (1998: 198), L. pseudoimbricata Levkov, Metzeltin & A.Pavlov (2013: 193) and L. wetzelii. The new species can be distinguished by narrowly rounded apices and proximal raphe endings located close to each other, which in L. madagascarensis continue as irregular, shallow, elongated grooves. Also, the species mentioned above differ in their ecology, particularly their salinity preference: L. madagascarensis was found on mangrove roots in water with salinity of 15 PSU, while other species were reported from freshwater environments.

In L. nosybeana, the isolated pore openings from the exterior to the interior are displaced; externally, the isolated pore opening is located near the margin, while internally, the isolated pore opening is located halfway between the valve margin and valve center. This is a relatively distinctive feature of the species. The only other Luticola taxa showing this arrangement include L. voigtii (Meister) D.G.Mann in Round et al. (1990: 671) and L. galapagoensis Witkowski, Bak, Kociolek, Lange-Bertalot & Seddon in Bak et al. (2017: 203) – both tropical brackish-water species.

The genus Luticola is usually regarded as freshwater and has been frequently reported from inland waters and associated habitats (mostly aerophilous environments). Hustedt (1964) and Levkov et al. (2013) refer to (and describe) several species from brackish-water habitats, mostly in the tropics. It would be interesting to find out whether the ecological preferences of the species, and thus the distribution, are related to its phylogeny.

Finally, the species of Luticola described in this paper, for which we were able to observe the valve interior, i.e. L. nosybeana, is characterized by canals developed around the internal margins (see Fig. 4a–e), a feature common to most Luticola species described to date (e.g. Metzeltin et al. 2005; Levkov et al. 2013; Bak et al. 2017; Glushchenko et al. 2017; Straube et al. 2017). In the species from Madagascar, however, these canals are not well developed, and – as opposed to other Luticola taxa (e.g. L. uruguayensis Metzeltin, Lange-Bertalot & García-Rodrígez, 2005: 118) – not conspicuous on one side of the central area. Further research on this interesting genus is necessary to understand whether the difference in the size of the marginal canals, as well as the distinctive external proximal raphe endings present in the two new species described from Madagascar have ecological, biogeographic and/or phylogenetic significance.

The study involved materials collected from mangrove roots at the coast of Nosy Be, a virtually unexplored tropical island off NW Madagascar. The materials contained numerous unidentified taxa from exposed habitats, including Humidophila and Luticola. Two taxa of Luticola described here, L. nosybeana and L. madagascarensis, are new to science, which was confirmed by LM and SEM examination and comparisons with already known taxa. The comparisons show that the newly described species are different in terms of their morphology (valve shape, raphe structure, isolated pore location and structure) and dimensions. Importantly, they also differ in terms of habitat, with L. nosybeana andL. madagascarensis inhabiting the mangrove roots of brackish waters, whereas all similar taxa live in freshwaters. Research on the mangrove root materials is ongoing and aims to explore the whole diversity of diatoms of this important habitat.