Nodosilinea chupicuarensis Vázquez-Martínez, Gutierrez-Villagomez and Molina-Torres, 2018

Vázquez-Martínez, Juan, Gutierrez-Villagomez, Juan Manuel, García, Citlali Fonseca-, Ramírez-Chávez, Enrique, Mondragón-Sánchez, Ma. Lourdes, Martínez, Laila Partida-, Johansen, Jeffrey R. & Molina-Torres, Jorge, 2018, Nodosilinea chupicuarensis sp. nov. (Leptolyngbyaceae, Synechococcales) a subaerial cyanobacterium isolated from a stone monument in central Mexico, Phytotaxa 334 (2), pp. 167-182 : 171-175

Nodosilinea chupicuarensis Vázquez-Martínez, Gutierrez-Villagomez and Molina-Torres sp. nov. ( Fig. 2 and 3 )

Diagnosis: —Differing from all other described Nodosilinea species through possession of regularly spiralled filaments in the population.

Description: —Thallus creeping on the rock surface forming a blue-green patina, embedded in mucilage. Filaments long (up to more than 350 cells long), under high light straight or gently curved, uniseriate, without false branching, 1.1–1.3 μm wide, under low light intensity (<4 μmol photons m 2 s-1) appearing curved and forming spirals, in some instances filaments multiseriate, occasionally forming tight, compact nodules. Sheaths clear, thin, occasionally extending beyond trichome ends. Trichomes slightly motile, particularly at the 10–20 cells at the tips, constricted at the cross-walls, 0.9–1.2 μm wide. Cells lacking aerotopes, barrel-shaped (0.9 μm wide x 1.2 μm long), occasionally discshaped (1.2 μm wide x 0.6 μm long) or isodiametric (0.9–1.2 μm in diameter), with clear centroplasm and chromoplasm often visible in LM (=peripheral thylakoids). Apical cells dome-shaped, non-capitate, sometimes elongated, without calyptra. Reproducing by motile hormogonia.

Etymology: — Nodosilinea chupicuarensis , Nodosilinea = “Knotted line” Perkerson et al. (2011); chupicuarensis (chu.pi.cua.re´n.sis) N.L. fem. adj. chupicuarensis = origin from Chupícuaro, the ancient name of the region where the type strain was isolated (from Purépecha “ chupicua ” the name of a plant of the Impomea genus used to obtain a blue stain combined with “ ro ” meaning “place”; consequently chupicuaro = “blue place”).

Habitat: —Stone surface, epilithic.

Type Locality:— MEXICO. Guanajuato: San Miguel de Allende, archaeological zone of Cañada de la Virgen, Complex A, 20°51’29.6” N, 100°55’41.0” W, J. Vázquez-Martínez, J.M. Gutierrez-Villagomez, J. Molina-Torres, 12- 10-2012.

Holotype here designated: — MEXU 4813 ! in the Herbario Nacional at the Universidad Nacional Autónoma de México at Mexico City, Mexico.

Isotypes here designated: — MEXU 4814!, MEXU 4815! in the Herbario Nacional at the Universidad Nacional Autónoma de México at Mexico City, Mexico.

Observations: —Under low light intensity conditions (3–4 weeks of culture), filaments are curved to form spirals that can be very compact or very relaxed. In some instances, trichomes become multiseriate due to cells inside the filament dividing in multiple planes. In some cases, these multiseriate regions resemble nodules. Within these multiseriate regions, nodules occasionally occur with the characteristic morphology of nodules found in other Nodosilinea species. This strain presents a distinctive spirally coiled morphology under the same low light intensity conditions that produce nodules in other species, with only rare nodule formation.

The thallus is formed by a cluster of filaments that becomes a firm mat. In some instances, the filaments are embedded in an extracellular polymeric matrix, and these polymers can be visualised by alcian blue staining (data not shown). The thallus grows as a cluster of tangled filaments that are inlaid in the rock porosities forming a green-blue patina. Something similar occurs in vitro when the strain grows in solid BG-11 medium. Trichomes grow as spherical compact aggregates (1–4 mm diameter) when the strain is cultivated in liquid BG-11 medium under constant agitation. Hormogonia are formed by direct trichome fragmentation without formation of necridia.

Phylogenetic analysis: —According to the phylogenetic analysis using 16S rRNA gene sequences, Nodosilinea chupicuarensis is most related to Nodosilinea nodulosa UTEX 2910 , with 99.5–99.4% identity. In the 16S rRNA phylogenetic tree ( Fig. 4 ), N. chupicuarensis is the sister taxon to N. nodulosa UTEX 2910 and these two species are grouped together in a weakly supported clade in all three analyses performed (posterior probabilities/bootstrap support = 0.68, 76, 70). However, based on the analysis of the 16S-23S ITS region, N. chupicuarensis was distant from Nodosilinea nodulosa UTEX 2910 (p-distance = 0.0822), being closest to a soil strain from China, Nodosilinea sp. CXA007.4 (p-distance = 0.0665) (see Table 1). Members of the same bacterial species have been found to have p-distance <0.03, and the average intraspecies p-distance is ~0.01 ( Erwin & Thacker 2008, Osorio-Santos et al. 2014, Pietrasiak et al. 2014), strongly suggesting that N. chupicuarensis is distinct from all other sequenced species (described or undescribed) of Nodosilinea . The 16S-23S ITS phylogenetic tree ( Fig. 5 ) shows N. chupicuarensis to be phylogenetically separated from the other Nodosilinea strains, which fall into one of two supported clades.

ITS secondary structure: —Comparison of secondary structure of 16S-23S conserved domains provides additional confirmation of the phylogenetic similarity among all Nodosilinea species presented in this study. While the secondary structure among the D1–D1’ helices is identical for six of the strains including N. chupicuarensis ( Fig. 6 A–F ), every helix differs in sequence ( Fig. 6 A–H ). The Box-B helices were more variable in length, sequence, and structure ( Fig. 6 I–P ), and could be considered evidence that all eight strains in the comparison set were likely different species. Unlike most other cyanobacterial genera, the V 3 helices were unusually conserved. All 19 strains for which complete ITS regions were obtained had V 3 helices identical in sequence and structure ( Fig. 6 Q ).

Figures

FIGURE 2. Main characteristics of Nodosilinea chupicuarensis. A-C. Mature filaments forming loose to tight spirals. D. A characteristic nodule (arrow). E. Multiseriate filament. F. Filaments with mature, elongated end cells (arrows). All figures to same scale, scale = 10 μm.

FIGURE 3. SEM micrographs of Nodosilinea chupicuarensis.A. Mature uniseriate filament forming a tight spiral. B. Amplification of a spiral. C. Coiled filaments.

FIGURE 4. Phylogenetic position of Nodosilinea chupicuarensis (denoted with an arrow) in a Bayesian Analysis (285 OTUs, 1222 positions), with support values on nodes representing BA posterior support/ML bootstrap support/MP bootstrap support, respectively. The uncollapsed tree from which this figure was derived appears in supplemental materials.

FIGURE 5. Unrooted tree of Nodosilinea strains based on Bayesian Analysis of 19 sequences (562 DNA positions, 114 coded indels). Bootstrap values from the parsimony analysis (562 DNA positions, gaps coded as a fifth base) are mapped onto the nodes in which the analyses were in agreement.

FIGURE 6. Secondary structure of conserved domains of the 16S-23S ITS regions for 8 representative strains of Nodosilinea. A–H: D1- D1’ helices; I–P: Box-B helices; Q: V3 helix (all 19 strains). Strain labels in the bottom row apply to the D1-D1’ helices in the first row. Bases differing from those in N. chupacuarensis are indicated by hollow circles next to the differing bases.