Phenological segregation suggests speciation by time in the planktonic diatom Pseudo-nitzschia allochrona sp. nov

Abstract

The processes leading to the emergence of new species are poorly understood in marine plankton, where weak physical barriers and homogeneous environmental conditions limit spatial and ecological segregation. Here, we combine molecular and ecological information from a long-term time series and propose Pseudo-nitzschia allochrona, a new cryptic planktonic diatom, as a possible case of speciation by temporal segregation. The new species differs in several genetic markers (18S, 28S and ITS rDNA fragments and rbcL) from its closest relatives, which are morphologically very similar or identical, and is reproductively isolated from its sibling species P. arenysensis. Data from a long-term plankton time series show P. allochrona invariably occurring in summer-autumn in the Gulf of Naples, where its closely related species P. arenysensis, P. delicatissima, and P. dolorosa are instead found in winter-spring. Temperature and nutrients are the main factors associated with the occurrence of P. allochrona, which could have evolved in sympatry by switching its phenology and occupying a new ecological niche. This case of possible speciation by time shows the relevance of combining ecological time series with molecular information to shed light on the eco-evolutionary dynamics of marine microorganisms.

Keywords: cryptic species; diatoms; eco‐evolutionary dynamics; long‐term ecological research (LTER); phenology.

FIGURE 1

Morphology of Pseudo‐nitzschia allochrona sp. nov. LM (a, b) and TEM micrographs (c–j). (a) Cells in girdle view, strain MC784 4 II. (b) Long cells formed following sexual reproduction (cross of strains 9A2x9C3A, Table S3). girdle view. (c) Whole valve. (d) Valve end. (e) Valve end. (f) Central part of the valve face with central nodule. (g) Central part of the valve with central nodule and mantle (arrowheads). (h) Detail of the valve striae with two rows of pores typical of the P. delicatissima‐complex. (i) Detail of the valvocopula. (j) the three cingular bands, with arrows indicating their borders: V = valvocopula, II = second cingular band, III = third cingular band. (a): Strain MC784 4 II, (b): (c–j): Strain SZN‐B109. Scale bars: (a) = 5 μm, (b) = 20 μm, (c) = 10 μm, (d–f) = 1 μm, (g, h, j) = 0.5 μm, (i) = 0.2 μm.

FIGURE 2

Life stages of Pseudo‐nitzschia allochrona sp. nov. during sexual reproduction. LM (a, f) and SEM (b–e). (a) Two paired gametangia of opposite mating types and different sizes, cross of strains 9A2x9C5. (c) Gametangia with two zygotes connected to the parental valve, cross 9B4x9C5. (c) Early auxospores, cross 9B4x9C5. (d) Elongated auxospores, cross 9B4x9C3a. (e) Mature auxospores with a bulge in the center (arrowheads), still connected to the parental valve, cross 9B4x9C5. (f) Long cell following the first divisions with a distinct central bulge (arrowhead), cross 9A2x9C5. Scale bars: (a and f) = 20 μm, (b) = 5 μm, (c–e) = 10 μm.

FIGURE 3

Maximum‐likelihood phylogenies of the P. delicatissima‐complex species living in sympatry in the Gulf of Naples. Excerpts from Figure S1 representing the complete phylogenetic trees. (a) 18S; (b) 28S; (c) ITS; and (d) rbcL. The new species P. allochrona is well separated in all markers from the closely related species that occur in the Gulf of Naples, namely, P. delicatissima, P. dolorosa, and P. arenysensis, and it is sister to P. arenysensis in all supported phylogenies, being closer to P. delicatissima only in the non‐supported 18S phylogeny.

FIGURE 4

ITS2 secondary structure of P. allochrona (SZN‐B509) and comparison to that of the sibling species P. arenysensis (NerD1). Dashed black boxes: Indels in the alignment of the two species; red boxes: CBCs; green boxes: Hemi‐CBCs; yellow boxes: SNPs; and dashed purple box: A hemi‐CBC generating an internal pyrimidine‐pyrimidine loop (+). Red circles indicate intraspecific ITS polymorphisms between P. allochrona strains from the Gulf of Naples and those from the Ionian Sea (SZN‐B495).

FIGURE 5

Distribution and ecology of the species of the P. delicatissima‐complex in the Gulf of Naples. (a) Annual distribution (1984–2015) of P. allochrona and other P. delicatissima‐like species (P. delicatissima, P. arenysensis, and P. dolorosa, lumped), light microscopy data. Lines represent different years. P. Allochrona was distinguished from its cryptic congeneric species based on its recurring occurrence in summer–autumn. The exceptional peak in late June–early July 2014 (dashed line) was not attributable to either species and hence was not included in the niche analysis of panel d. (b) Separation of P. delicatissima‐like species in the seasonal space identified by day length and temperature values. Letters are month names' initials. (c) Annual distribution of P. allochrona and the three other P. delicatissima‐like species living in the Gulf of Naples based on the isolation date (2004–2016) of 62 and 187 molecularly identified strains, respectively. (d) Niche analysis showing P. allochrona separated from the other congeneric species along the OMI axis 1, highly correlated with temperature (temp) and negatively correlated with nutrients (DIN): Dissolved inorganic nutrients; phos: phosphate; si: silicate. More and larger dots in the 3rd and 4th quadrants indicate higher frequency and abundance of all species with greater day length (day l) and lower salinity (Sal). Gray dots are samples with no P. delicatissima‐like species. (e) Interannual density variations (lines: annual average values; shadowed areas: CI 95%) of P. delicatissima‐like species at the LTER‐MC site in the Gulf of Naples.