Reproduction in Haptophytes

Haptophytes, a group of unicellular algae, are widely distributed and abundant in various marine environments. While most haptophytes exist as solitary, motile or non-motile forms, some can form colonies or short filaments. Haptophyte cells are typically covered with one or more layers of organic scales that are formed within vesicles derived from the Golgi apparatus. A distinguishing feature of haptophytes is the presence of a unique organelle called a haptonema, which resembles a flagellum but has distinct differences in microtubule arrangement and function. The haptonema is thought to be involved in attachment or capture of prey, and is present in most species, although it may be reduced or absent in some rare cases.

 

The Haptophyta phylum is divided into two classes: the Pavlovophyceae, which has only 13 known species, and the Prymnesiophyceae, which encompasses the majority of haptophyte diversity. The Prymnesiophyceae class is further subdivided into two orders of non-calcifying taxa, namely the Phaeocystales and the Prymnesiales, along with the calcifying coccolithophores, which form a monophyletic clade known as the sub-class Calcihaptophycidae. This sub-class includes four orders: Isochrysidales, Coccolithales, Syracosphaerales, and Zygodiscales.

 

Some well-known haptophyte taxa, such as Phaeocystis, Prymnesium, and Chrysochromulina, are non-calcifying and are known to form periodic harmful or nuisance blooms in coastal environments. However, the most recognized haptophytes are the coccolithophores, which are members of the Prymnesiophyceae class. Coccolithophores have both a proximal layer of organic body scales and a distal layer of calcified scales called coccoliths, which are also formed intracellularly and often exhibit intricate ornamentation. Coccolithophores play a crucial role in global carbon cycling as they are responsible for a significant portion of modern oceanic carbonate production (Rost and Riebesell 2004).

 

Many members of the haptophyte class Prymnesiophyceae exhibit heteromorphic life histories, with documented alternations between non-motile and flagellated stages, colonial and single cell stages, as well as benthic and planktonic stages. Early studies on haptophyte life cycles focused primarily on coccolithophores from the families Pleurochrysidaceae and Hymenomonadaceae (order Coccolithales), as they were relatively easy to maintain in laboratory culture. Alternation between a non-calcifying stage (referred to as 'Apistonema') and a coccolith-bearing stage has been observed in species such as Ochrosphaera (Schwarz 1932; Lefort 1975), Hymenomonas (Fresnel 1994), and Pleurochrysis (Leadbeater 1970, 1971; Gayral and Fresnel 1983). In Pleurochrysis carterae (Rayns 1962) and Hymenomonas lacuna (Fresnel 1994), chromosome counting has confirmed that the non-calcifying stage in these life cycles is haploid, while the calcifying stage is diploid, providing solid evidence of the existence of haplodiplontic life cycles in haptophytes (Fig. 6).

Figure 6

The presence of coccoliths, which are visible in light microscopy, made it relatively easy to discern the different life cycles. There are two main types of coccoliths: heterococcoliths, which are composed of a radial array of interlocking crystals with complex shapes, and holococcoliths, which are constructed of numerous small, similarly sized and simple-shaped calcite elements. A culture study conducted by Parke and Adams (1960) on the non-motile stage of Coccolithus braarudii (Coccolithales) bearing heterococcoliths demonstrated an alternation with Crystallolithus hyalinus, a motile stage bearing holococcoliths. Prior to this observation, heterococcolithophores and holococcolithophores had been considered as distinct taxonomic groups of species.

 

Billard (1994) reviewed various studies and proposed that haptophyte life cycles typically involve both haploid and diploid phases capable of independent asexual reproduction, known as haplodiplonty. These phases are characterized by distinct patterns of body scale ornamentation, and in some cases, coccolith type. Body scales of Prymnesiophyceaen species are composed of microfibrils and contain proteins and carbohydrates, including cellulose (Leadbeater 1994 and references therein). The proximal ("body") scales consist of two layers, with the proximal face, facing the cell membrane, showing a radial pattern of microfibrils often arranged in quadrants. The distal face, on the other hand, exhibits either a radial or an interwoven spiral pattern of concentric rings.

 

In Billard's scheme, the body scales of diploid cells exhibit identical radial ornamentation on both sides, while those of the haploid stage display distinct patterns on the proximal and distal faces, with radial and spiral patterns, respectively. Based on the patterns of body scale ornamentation in known haplodiplontic life cycles of species in the Pleurochrysidaceae and Hymenomonadaceae, as illustrated by Manton and Leedale (1969), Billard (1994) predicted that the heterococcolith-bearing phase of C. braarudii is diploid, while the holococcolith-bearing phase is haploid. This pattern also fits with other coccolithophores, such as Syracosphaera pulchra (Inouye and Pienaar 1988), Umbilicosphaera hulburtiana (Gaarder 1970), and Jomonlithus littoralis (Inouye and Chihara 1983) for heterococcolithophores, and Calyptrosphaera sphaeroidea (Klaveness 1973) and Calyptrosphaera radiata (Sym and Kawachi 2000) for holococcolithophores, where body scales have been illustrated in only one phase.

 

DNA quantification using flow cytometry later confirmed the haplodiplontic nature of the life cycle of Coccolithus braarudii, as well as two other species where both phases were maintained in culture, as reported by Houdan et al. (2004). Additional indirect evidence supporting the widespread occurrence of haplodiplontic life cycles in coccolithophores comes from field observations of "combination coccospheres" bearing both heterococcoliths and holococcoliths, which have been interpreted as capturing the moment of a life cycle phase change. These observations have been reported from various locations by Thomsen et al. (1991), Kleijne (1992), Alcober and Jordan (1997), Young et al. (1998), Cros et al. (2000), Cortes and Bollmann (2002), Geisen et al. (2002), Cros and Fortuno (2002), indicating that life cycles with alternating heterococcolith-bearing (diploid) and holococcolith-bearing (haploid) stages are widespread across the diversity of coccolithophores.

 

Culture studies and observations of combination coccospheres have provided evidence that diploid generations in coccolithophore life cycles consistently bear heterococcoliths, while haploid generations are covered by various types of structures such as holococcoliths (Coccolithaceae, Calcidiscaceae, Helicosphaeraceae, Syracosphaeraceae), aragonitic coccoliths (Polycrater), nannoliths (Ceratolithaceae), a non-calcifying benthic stage (Pleurochrysidaceae, Hymenomonadaceae), or a non-calcifying motile stage (Noëlhaerhabdaceae), as reported by Billard and Inouye (2004) and references therein. This suggests that there are distinct characteristics associated with diploid and haploid stages in coccolithophore life cycles, which can be identified based on the types of structures they bear.

 

Each of the other two non-calcifying orders within the Prymnesiophyceae also exhibit alternation between generations of different ploidy levels. In the Phaeocystales, the life cycle of Phaeocystis globosa has been extensively studied (reviewed by Rousseau et al. 2007). It has been described as a haplo-diplontic life cycle, involving diploid colonial cells (either free non-motile cells or within colonies), diploid flagellate cells without organic scales, and two types of haploid flagellates surrounded by organic scales, differing in size (meso- and microflagellates). P. Antarctica is believed to have a similar life cycle (Zingone et al. 2011). In the Prymnesiales, Prymnesium parvum has been identified as the diploid stage in a life cycle where the haploid stage was initially described as a separate species, P. patelliferum (Larsen and Medlin 1997; Larsen and Edvardsen 1998). Prymnesium polylepi (=Chrysochromulina polylepis) has also been shown to have a haplo-diploid cycle (Edvardsen and Vaulot 1996; Edvardsen and Medlin 1998). In each of these cases, the body scale ornamentation conforms to the scheme proposed by Billard (1994), and in some cases, morphological differences are also evident in the distal organic scales (e.g., Probert and Fresnel 2007). However, the details of these non-calcified scales can only be observed using electron microscopy, which can make the identification of life cycles in these non-calcifying haptophytes challenging.

 

In summary, dimorphic haplo-diplontic life cycles seem to be widespread in the Prymnesiophyceae. So far, alternation of generations has not been demonstrated in members of the other haptophyte class, the Pavlovophyceae. Unlike the Prymnesiophyceae, species within the Pavlovophyceae do not possess the ornamented plate scales that have often been indicative of ploidy state, and thus, the potential existence of haplo-diplontic life cycles in the Pavlovophyceae has not been recognized. Transitions from non-motile to motile cells are common in the Pavlovophyceae (Billard 1994; Bendif et al. 2011), but relative motility is not always a reliable indicator of ploidy state.

 

There are limited reports of cysts in the Haptophyta. Cysts of Prymnesium were described by Carter (1937) and Conrad (1941) and have been studied by Pienaar (1980), who showed that the walls of Prymnesium parvum cysts consist of layers of scales with siliceous material on the distal surfaces. It is currently unknown whether the formation of these cysts is related to the ploidy level of the species.

 

It is noteworthy that the existence and role of sexuality, if present, in haptophyte life cycles remains largely unknown (Billard 1994). In coccolithophores, sexuality has been directly observed in only three species: Ochrosphaera neapolitana (Schwarz 1932), Pleurochrysis pseudoroscoffensis (Gayral and Fresnel 1983), and Coccolithus braarudii (Houdan et al. 2004). In O. neapolitana, Schwarz (1932) reported meiosis, isogamete formation, and syngamy. Insights into the meiotic process in coccolithophore life cycles can be inferred from light microscope observations of Coccolithus braarudii (Houdan et al. 2004) and Pleurochrysis pseudoroscoffensis (Gayral and Fresnel 1983). In Coccolithus braarudii, meiosis may occur within the heterococcosphere prior to the production of the flagellar apparatus, and the resulting motile cell that emerges may be diploid, with nuclear reduction occurring in subsequent divisions. This pattern, where only one viable cell emerges, is similar to meiosis in the chlorophyte Spirogyra (Harada and Yamagishi 1984). Alternatively, the motile cell that emerges from the heterococcosphere may still be diploid, and nuclear reduction occurs in subsequent divisions. The observation that holococcolith production does not immediately commence after the formation of the flagellar apparatus supports this second hypothesis (Houdan et al. 2004). In the life cycle of P. pseudoroscoffensis, four non-calcified motile haploid cells are formed within a heterococcosphere, and upon release, these cells remain motile briefly before settling and dividing asexually to initiate the haploid non-calcified pseudofilamentous stage (Gayral and Fresnel 1983). Comparable "meiospores" are formed in the life cycles of other members of the Pleurochrysidaceae (von Stosch 1967; Leadbeater 1971) and the Hymenomonadaceae (Fresnel 1994). The mode of initial gamete attraction and contact in coccolithophores is not known, but once initiated, syngamy can be completed rapidly within a very short period of time. The rapid initiation of heterococcolith production in the zygote observed in C. braarudii by Houdan et al. (2004) and reported for P. pseudoroscoffensis by Gayral and Fresnel (1983) indicates that the two haploid nuclei must fuse immediately following cytoplasmic fusion. In both species, a complete heterococcosphere was formed within 24 hours of the onset of fusion. Based on this limited evidence, it appears that fusing gametes in coccolithophores are isogamous and morphologically indistinguishable from vegetative haploid cells, and that fusion can occur within a clone (homothallism). To the best of our knowledge, crossing experiments between haploid coccolithophore strains have not been attempted.

 

Phaeocystis globosa exhibits a complex life cycle wherein micro- and mesoflagellates (haploid stages) are produced within the colony, presumably through meiosis, and eventually released to multiply vegetatively. The life cycle is believed to be completed by syngamy between a micro- and mesoflagellate, resulting in the formation of a diploid macroflagellate that may develop into a new colony. However, the formation of colonial stages has not been observed in cultures containing only haploid cells of P. globosa (Vaulot et al. 1994). This could be explained by the possibility that conjugation only occurs between the two heteromorphic types of haploid flagellates (anisogamy) or that different mating types exist among the haploid flagellates (heterothallism). In P. Antarctica, a non-motile zygote that links the haploid unicellular stages and the diploid colonial stages has been documented, which can divide vegetatively as a benthic palmelloid stage and may not revert to the colonial stage, at least under culture conditions (Gaebler-Schwarz et al. 2010).

 

The study of sexual reproduction mechanisms in haptophytes is currently constrained by the limited availability of cultures and the lack of clear understanding of the factor(s) responsible for inducing life cycle phase changes in this group. The transition between life stages in haptophytes is believed to be controlled by a combination of endogenous and environmental factors, but their role and relative importance remain poorly understood. Several reports suggest that factors such as temperature, light (Leadbeater 1970), and the addition of fresh medium (Inouye and Chihara 1979; Gayral and Fresnel 1983) may influence phase changes in Pleurochrysidaceae. In cultures of Calyptrosphaera sphaeroidea, Noel et al. (2004) demonstrated that exposure to specific vitamins and trace metals induced the transition to the heterococcolith-bearing phase, while slightly higher concentrations of components in the basic medium along with concomitant stresses of light and temperature induced the formation of the holococcolith-bearing phase. However, due to the limited number of cultures available and the complex interplay of various factors, our understanding of sexual reproduction in haptophytes remains incomplete.

 

Houdan et al. (2004) proposed that concentrations of inorganic or organic trace elements in the medium may play a role in inducing phase changes in three coccolithophore species, and also hypothesized the involvement of a biological clock in this process.

 

Life cycle transitions, with each phase adapted to distinct ecological niches, may be an integral part of the ecological strategy of haptophytes. In ecological terms, a Haplo-diplontic life cycle is generally considered an adaptation to environments that are seasonally variable or contain two different niches (see review by Valero et al. 1992). Evidence suggests that heterococcolith-bearing and holococcolith-bearing or non-calcifying phases of certain coccolithophore species have differential in situ spatiotemporal distributions (Cros and Fortuno 2002; Frada et al. 2012). Generally, holococcolith-bearing stages (which always possess flagella) may be adapted to warm stratified surface waters, while the more robust (and often non-motile) heterococcolith-bearing stages may be better suited to turbulent mixed-layer waters. Noel et al. (2004) extrapolated from growth medium preferences of the two stages of Calyptrosphaera sphaeroidea to propose a hypothetical ecological cycle in which the holococcolith-bearing stage occurs in offshore waters and the heterococcolith-bearing stage in coastal waters. Very few studies have experimentally compared the physiological characteristics of different ploidy stages in haptophytes, but consistent differences in responses to physical-chemical and/or biotic parameters have been found in these studies.

 

For instance, in the highly abundant coccolithophore Emiliania huxleyi, which has a life cycle with diploid non-motile heterococcolith-bearing cells alternating with haploid flagellate non-calcifying cells, the haploid stage appears to be relatively sensitive to high light (Houdan et al. 2005) but not susceptible to E. huxleyi-specific viruses (EhVs) that routinely infect and kill diploid cells (Frada et al. 2008). Dramatic differences in gene expression between diploid and haploid phases of E. huxleyi have been demonstrated, with greater transcriptome richness in diploid cells suggesting they may be more versatile for exploiting a diversity of rich environments, whereas haploid cells appear to be intrinsically more streamlined (Von Dassow et al. 2009).