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2. Phylogeny, Morphological & Genetic Features

2.a Phylogeny of C.taxifolia: The tropical strain of Caulerpa taxifolia is widespread among the tropics; with native populations found in the Atlantic Ocean (West Indies and African coast), the Indian Ocean (Pakistan, Sri Lanka, and north western Australia), and the Pacific Ocean (Philippines, Indonesia, Japan, New Caledonia, and north eastern Australia) where it grows in small patches and does not present problems. This marine, green alga, was first described by M.Vahl in 1802 as Fucus taxifolius and was regrouped in 1817 by C.Agardh into the following taxonomic position:


KINGDOM Protista (Gk. protos, first) by Raven et al., 1992 or
     Plantae (L. plant); by Postlethwait & Hopson, 1995 & 2.0ITIS, 2004
DIVISION / PHYLUM Chlorophyta (Gk. chloros, green + phyto, plant)
CLASS Ulvophycea (L. ulva, sedge + phyto, plant);
     Chlorophyceae according to 2.0ITIS, 2004
ORDER Caulerpales (L. caulis, stalk) referring to the stalk of the algae;
     Bryopsidales (Gk. bryon, moss + obsis, appearance;
       unicellular & multinucleate thalli) according to 2.0ITIS, 2004
FAMILY Caulerpaceae (Gk kaulos, stalk, thallus or stipe)
GENUS Caulerpa (Gk. herpo, to creep)
SPECIES taxifolia (L. taxi, yew-like + folia, leaves)
     referring to the dark-green and flattened foliage


Fig.2.a Taxonomic position of C.taxifolia (140kB)

The Chlorophyta (green algae) groups unicellular or multicellular photosynthetic organisms characterized by the presence of chlorophyll a and b, as well as various carotenoids. The carbohydrate food reserve is starch, which is stored in plastids. There are about 7000 known species (2.1Raven et al., 1992).
The Ulvophyceae is one of the 3 classes currently recognized in the chlorophyte lineage, the others being Chlorophyceae and Trebouxiophyceae (formerly Pleurastrophyceae) and is made up of approximately 1100 species (2.2Dumay et al., 2002) clustered among 100 genera. Most of which are found in temperate and tropical marine environments, and the majority of green "seaweeds", including well-known species of besides Ulva and Acetabularia besides Caulerpa, are placed in this class as well. Conversely, the Chlorophyceae and Trebouxiophyceae, and the Streptophytes, consist almost entirely of non-marine organisms. In classifications based on morphology and ultrastructure, the Ulvophyceae have been separated from other chlorophytes mostly on the basis of characters associated with mitosis, cytokinesis, and the flagellar apparatus of zoospores and gametes. In molecular analyses, however, the relationships among these three classes are less clear (2.3Mandoli et al., 2002). Moreover, the "siphonous" orders of Ulvophyceae (Cladophorales, Dasycladales, Caulerpales) are difficult to resolve vis--vis each other and with other Ulvophyceae (orders Ulotrichales and Ulvales); phylogenetic trees based on single gene sequences reveal long branch lengths between "siphonous" (Gk. tube), sequences and those of other chlorophytes (2.3Mandoli et al., 2002).
Green algae differ from higher plants by the absence of true organs such as roots, leaves, stems, flowers, and seeds. While true veins and cross walls are absent in the assimilators, they have fibrous tissue that provides enough support to allow Caulerpa to withstand changes in osmotic pressure and maintain its shape. A cuticle and a cell wall surround the slimy cytoplasm that is rich in organelles (2.3aDebelius & Baensch, 1997).
Members of the order Caulerpales contain a vast array of polymorphic marine (sub-)tropical macrophytes (most naturally occurring species are found between 10° and 30° latidude) with multinucleate siphonous forms (2.3aDebelius & Baensch, 1997), without cross-walls or segregative division, filled with numerous plastids (e.g. amyloplasts which is an unpigmented plastids for starch storage). Thus forming one huge cell, which is vulnerable to substantial plasma loss. Caulerpales though, are equipped with efficient wound healing properties (upon damage, healing occurs in seconds, involving actin-mediated contraction and a "plug" of cell wall material). The skeletal constituent of the cell wall contains xylan rather than cellulose and is reinforced with a multiaxial construction, and sometimes even stabilized with calcium carbonate deposits as in Halimeda sp.(2.4Strassburger, 1998). The thallus among members of the genus Caulerpa - in which approximately 75 species are currently recognized (2.3aDebelius & Baensch, 1997) - is non-septated but siphonous in structure and reinforced with anastomosing strands of wall material (trabeculae) which are ingrowths of the cell wall. A thin layer of cytoplasm, containing countless numbers of each type of organelles, is appressed to the wall (2.5Silva, 2002). Division of labour is achieved between photosynthetic chloroplasts and starch-storing leucoplasts (heteroplastidy).
Pyrenoids are other organelles found therein. These particles have strong light refracting properties and are found in the chloroplasts of many algae. The chloroplasts, which are usually located along the cell wall, capture energy from light (E = hν). Varieties that grow at greater depths often compensate for the shortage of light by increasing their photosynthetic surface area (2.3aDebelius & Baensch, 1997).
Furthermore, Caulerpa possess siphonaxanthin and siphonein as photosynthetically active pigments (2.5Silva, 2002). This alga also exhibits some particular biological and physiological features: resistance to low temperature, gigantism of the thallus, high growth rate, which differentiate it morphologically from the tropical strain (2.6Meinesz et al., 1995). Indeed, species in this genus are strongly varying, depending on the environment. Transplanting them from into a location where environmental factors deviate from conditions the plant was originally growing, unavoidably induces an adaptation or altered growth form whereby the original morphotype soon becomes unrecognizable (2.3aDebelius & Baensch, 1997).

2.b Morphology of C.taxifolia: Although individual plants are composed of only one cell, Caulerpa has a complex morphology, composed of pseudo-organs that often resemble the roots, shoots and leaves of higher plants. It is one of the most distinctive genera of seaweeds, making it identifiable solely on the basis of its habit (2.5Silva, 2002). It consists of a creeping rhizome that produces tufts of colourless rhizoids downward and photosynthetic branches (assimilators) upward. When dealing with dimensions, the aquarium strain is different in size, length, growth rate and temperature tolerance from samples collected in tropical areas (2.7Boudouresque et al, 1995). C.taxifolia in the coastal areas of tropical North to Central QLD and from Port Hacking has a delicate morphology with narrow stolons, fronds, and pinnules (2.8Benzie et al., 2000). In contrast, the aquarium strain of C.taxifolia in the Mediterranean is generally quite large with broader stolons, fronds, and pinnules, although variable depending on depth (larger in low light conditions) and season (2.6Meinesz et al., 1995). However, populations of C.taxifolia in southern Queensland, at Stradbroke Island, which experience environmental conditions more akin with the Mediterranean, express a range of morphologies that include plants with heavy stolons and long, broad fronds bearing large pinnules (2.8Benzie et al., 2000).
As a result, the shape and length of the fronds and internodes, the length of the horizontal axis, and the overall turgor of this algae is also determined by the substrate. When C.taxifolia grows on piles of rock close to the water surface (obtaining max. irradiance), it develops a short, stout form with heavy, thick rachis and stolons. The elongate looser form with more upright fronds is formed as it creeps over stones until a more suitable bottom substrate is encountered. Once a softer bottom is found, the rhizoids become shorter, with broader and more finely branched "root hairs". In this way environmental conditions allow the rhizoids to garner nutrients and affix the plant to various substrates (2.3aDebelius & Baensch, 1997) - see Chapter-3, 3.c.Nutrient Dynamics.

Fronds: One of the more striking external features regard the dimensions of the vertical fronds or pinnae (L. feather, the large but divided leaf - see figure 2.b). Fronds may be quite short or even absent in shallower water (leaving only the stolons), but are longer in deeper water with prevailing low light conditions. In the tropical version, primary fronds are 2-15cm in length, while those of the Mediterranean strain range reach average frond heights of 25cm - ranging from 5cm in shallower water, to 40cm at depths of 15m, and even to 60-80cm at greater depths (2.6Meinesz, 1995). The increase in frond length, under conditions of competition with Posidonia oceanica likewise indicates the alga's property of adaptation (2.2Dumay et al., 2002). Starting from the base, each frond is made up of the midrib or rachis (Gk. backbone), which represents the prominent central axis. Frond density varies according to the season and ranges from 5100/m2 in September to 14000/m2 in April (2.6Meinesz et al., 1995).

Rachis (Gk. backbone) is the main axis of the leaf (frond), from which the pinnules arise.

Pinnules (L. small feathers) grow out of this midrib to give each frond the characteristic feather-like appearance. Ramificating or branching fronds originate from primary pinnules. Pinnule length typically measures 1cm while pinnule density per side varies from 4 to 7/cm of rachis length. The shape of the pinnules is usually up-curved, tapered at the ends, with some even bifurcated at the top ends (split in two). According to Meinesz (2.101995), pinnule spacing and length is light dependant.

Stolon (L. shoot): Regularly spaced fronds are attached to the horizontally running stolon and is also the origin of the adventitious rootless or rhizoids. After the winter, regrowth originates from old stolons that have survived - maximum stolon length is 2.8m, while the overall cumulative stolon length tends to stabilize around an equilibrium value of 230m/m2 (2.11Meinesz & Hesse, 1991).

Rhizoids (Gk. roots) are rootlike extensions that absorb water, food, and nutrients. Unlike in vascular plants, Caulerpales do not have a root system composed of root cap and root hairs. Instead, regularly spaced rhizoid pillars descend vertically from the stolons. A few centimeters long, these pillars branch up into extremely thin filamentous rhizoids, that based on the substrate, can form a felt-like net penetrating through the substrate and thus stabilizing it along with the algae (2.10Meinesz, 1995; 2.12 Chisholm et al., 1996).

Fig.2.b Morphology of C.taxifolia 2.6aCreese et al., 2004; (85kB)



Fig.2.c In Situ morphology of C.taxifolia (190kB)

The debate whether C.taxifolia is an ecomorphic variant (an ecad) of another species, C.mexicana (2.13Jaasund, 1977; 2.14Coppejans and Beekman, 1990; 2.15Coppejans and Prud'homme van Reine, 1992), or a well-defined species in its own right (2.16Calvert et al., 1976; 2.17Silva et al., 1987) is still unsolved. According to most accepted taxonomic criteria, ecomorphic variants of C.taxifolia can, under certain conditions, possess the morphological characteristics attributed to C.mexicana, and vice-versa (2.18Chisholm et al., 1995; 2.19Olson et al., 1998). In order to differentiate C.taxifolia from the others, it must be mentioned that C.taxifolia is not the only invasive species of this genus that has conquered the Mediterranean Sea. While C.taxifolia has been accidentally released from the public aquarium at Monaco, C.racemosa, C.scalpelliformis and C.mexicana are considered to be Lessepsian immigrants from the Red Sea (2.7Boudouresque et al., 1996; 2.20Piazzi et al., 1997).

Therefore a brief excursion helps to highlight the morphological differences between Caulerpa taxifolia, C.mexicana, C.scalpelliformis, C.racemosa and the autochthonous species C.prolifera.


Caulerpa mexicana: Partings of rhizoids are very closed on the stolon (<1cm). The pinnules are wide and short; the ratio length on width (L/w) ranges from 2.5 to 5, and is a function based on environmental conditions. Fronds reach an average length of 6cm. Minimal viable temperature is 16C.

Fig.2.d C.mexicana (85kB)

Caulerpa scalpelliformis: Straight fronds that are about 10-20cm long. It closely resembles C.taxifolia but can be distinguished by the form of its branches, which are curved towards the interior in the latter and straight in the former. The rachis of the fronds is quite dominant and thicker than the pinnules. Another feature that distinguishes it from C.taxifolia are its pointed pinnules.

Fig.2.e C.scalpelliformis (85kB)

Caulerpa racemosa: An algae with long stolons; its racemous-rhizoids point downward. Photosynthesis occurs in the clustered aggregations that, opposing the rhizoids, merely reach a few cm in height. Growth pattern of phtotosynthetic thalli can vary from bifurcated with tiny branches to entirely rounded bubbles. This algae is likewise extremely invasive in Mediterranean subtidal habitats and as with C.taxifolia causes major modifications to benthic communities (2.21Verlaque & Fritayre, 1994; 2.22Villele & Verlaque, 1995; 2.23Bellan-Santini et al., 1996; 2.24Ceccherelli & Cinelli, 1997; 2.25Piazzi et al., 2001). It is currently in the process of spreading into the western Mediterranean (2.26, 2.27Piazzi et al., 1994, 1997; 2.28Gambi & Terlizzi, 1998; 2.29Modena et al., 2000; 2.30Verlaque et al., 2000) and it seems to be capable of even out-competing C.taxifolia (2.31Ceccherelli et al., 2002).

Fig.2.f2.37 C.racemosa (85kB)

Caulerpa prolifera: This native alga has few but robust stolons with the branching blades protruding perpendicularly into the water in intervals of 1-2cm. The superior part of each blades house the assimilatory pigments. The blades can reach a length of up to 15cm and can in some cases be even 13cm wide. Usually they have an oval or linearly elongated appearance with a smooth border following a helical pattern towards the apex (2.37 Marcabruno-Gerola, 1968).


Fig.2.g C.prolifera (80kB)

2.c Genetic Features of C.taxifolia: Genetic and evolutionary processes are often key features in determining whether invasive species establish and spread. Invasive species offer an excellent opportunity to study rapid evolution, and some of the best-documented examples of this phenomenon have come from invasive species (2.38Sakai et al., 2001).
The DNA fingerprints of C.taxifolia presented here support existing evidence for the descent of the Mediterranean C.taxifolia from an aquarium strain. The introduction of C.taxifolia via the Oceanographic aquarium in Monaco is strongly supported on the basis of having identical internal transcribed spacer (ITS) rDNA sequences (2.32Jousson et al., 1998). The phylogenetic analysis of these sequences show that the Mediterranean alga is genetically identical to the strain cultivated in aquaria (see figure 2.h - left image). Interestingly, the aquarium strain differs from all tropical populations of Caulerpa in lacking internal transcribed spacers (ITS) polymorphism, a fact that can be related to a prolonged confinement under aquarium conditions.

This finding has been confirmed in a repetitive analysis performed by Jousson et al. (2.33 2000), that included strains extracted from the Californian site. Eleven out of 12 Californian sequences were found to be identical to all aquarium and most Mediterranean sequences (fig. 2.h-right). 64 sequences from the Mediterranean-aquaria-California isolates were identical, whereas five were slightly divergent (from 0.4 to 1.1%). The sequences reveal a relatively robust clade (80% bootstrap value) grouping Californian, Mediterranean, aquaria and some Australian sequences together. Thus, confirming that even the Californian species belong to the aquarium strain.

Fig.2.h Phylogenetic analysis of C.taxifolia (80kB)

Benzie et al., (2.342000) tested the genetic relationships of the fine and robust morphological types of C.taxifolia and assessed the extent of genetic variation among widely separated populations of aquarium C.taxifolia with similar morphology of those found in Australia. Furthermore it was tested if C.taxifolia could be an ecomorph of C.mexicana. The findings revealed that allele frequencies were markedly different between C.mexicana and C.taxifolia populations. A cluster analysis grouped all of the C.mexicana populations together and separately from all of the C.taxifolia populations (fig. 2.i). The genetic distances between these species were generally far greater than between populations of each species.
Although populations of C.taxifolia that can develop a phenotype more akin to C.mexicana (Chisholm et al., 1995), both allozyme analysis and ribosomal sequence data confirm the true genetic differences among them (2.35Olsen et al., 1998) and that there are indeed two separate taxa, however some populations of C.taxifolia may overlap in morphology with C.mexicana.

Fig.2.i Phylogenetic analysis of C.taxifolia (62kB)

Wiedenmann et al., (2.302001) compared samples from 11 locations in the Mediterranean Sea with 3 representatives from public aquaria. The uniformity of hybridization patterns indicates that representative specimens from the Mediterranean and aquaria belong to the same clone. The slight differences in hybridization patterns in C.taxifolia from Manly Harbour (Australia) suggest that it carries very similar chloroplast and mitochondria traits. The Australian population of Manly Harbour/Moreton Bay is well suited for comparative studies of the role of C.taxifolia in a non-Mediterranean ecosystem because of the close relationship to the aquarium strain.

The comparative results of C.taxifolia strains from aquaria, the Mediterranean Sea and from Manly Harbour (Australia) are shown in fig. 2.j in which (CAC)5-hybridised Southern blots of total DNA after TaqI digestion (restriction patterns in ethidium-bromide stained agarose gels) have been performed. In contrast, the control sample (left lanes 1-2) clearly distinguishes them from the aquarium strain (C.prolifera, C.taxifolia from Martinique). The aquaria strains from Stuttgart and Enoshima reveal identical restriction patterns as samples from the Mediterranean Sea (Monaco, Krk, Sicily, Mallorca, Elba 1-3, St.Cyprien 1-3). Only slight differences in the position of a single band (indicated by the circle) were detected between the sample from Manly Harbour (lane 16) and the aquaria specimens.


Fig.2.j Fingerprints of C.taxifolia (40kB)

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