An interactive key to the Chrysochromulina species (Haptophyta) described in the literature

Abstract We present a general overview of features and technical specifications of an original interactive key web application for the identification of Chrysochromulina species. The list of species, originally described as belonging in the genus Chrysochromulina, is given and recent taxonomic changes in species and genera of the order Prymnesiales are provided. We briefly discuss the interest of such a key for the identification of phytoplanktonic species.


Introduction
Th e genus Chrysochromulina, erected by Lackey (1939) is an important component of the marine and brackish phytoplankton although the type species occured in fresh water. Electron microscopy (EM) has been a key tool for a specifi c identifi cation and Mary Parke & Irene Manton were pioneers in reviewing the type species  and describing more than ten species between 1955 and 1966. Th ey remained for many years the specialists of the genus until Barry Leadbeater added some more new species so that almost half the number of species known today were described by 1974. With the extent of TEM (Transmission Electron Microscopy) or SEM (Scanning Electron Microscopy) studies, the genus appeared worldwide distributed and some species were found to produce massive blooms, some of which were eventually toxic (Moestrup 1994). Th e 1988 bloom of Chrysochromulina polylepis Manton & Parke (Dahl et al.1989) was the fi rst event of toxic bloom causing important economic impact, raising a considerable interest of the scientifi c community especially in Scandinavia. Two PhD thesis were submitted , Eikrem 1999 with an illustrated key for identifi cation of species of this genus, based on morphological characters . With the 21th century molecular biology introduced changes in the delineation of classes and orders and the genus Chrysochromulina was considered as polyphyletic (Edvardsen et al. 2000).
Th e class name Haptophyceae was fi rst used by Christensen in 1962 but Hibberd introduced the typifi ed class name Prymnesiophyceae (Hibberd 1976), both names being considered as valid. More recently Silva et al. (2007) advise the use of the name Coccolithophyceae for this class, considering that the class name Coccolithophyceae Rothmaler 1951 had priority over Haptophyceae and Prymnesiophyceae. However this class name remains a matter of debate and therefore is not mentionned in the title.
Within the class, the genus Chrysochromulina was for a long time placed in the order Prymnesiales and the family Prymnesiaceae. However, from DNA phylogenies and morphological comparisons, Edvardsen et al. (2011) (Edvardsen et al. 2011). An unnamed species, cited as Chrysochromulina sp4 (Eikrem & Edvardsen, 1999), is considered as the type species of the new genus Pseudohaptolina Edvardsen & Eikrem. Th ey give a formal description of this species as P. arctica Edvardsen & Eikrem (Edvardsen et al. 2011). Because of these changes, the family Chrysochromulinaceae Edvardsen, Eikrem & Medlin is now restricted to the unique genus Chrysochromulina with the remaining species, all being saddle-shaped cells (Edvardsen et al. 2011).
As we are dealing here with an identifi cation key, we have taken into consideration all species originally described as Chrysochromulina in the literature (or moved to this genus as for Chrysocampanula spinifera (Fournier) by Pienaar and Norris in 1979) but modifi cations of their taxonomic status are mentioned in the species descriptions. References are restricted to papers giving the original description of a species or an emended description.

Taxonomic coverage
Th e key covers 58 species originally described as Chrysochromulina . References for publications dealing with their description and occurrence are given. A detailed description is provided and illustrations of a whole cell as well as for the diff erent scale types, in some cases from unpublished material seen in SEM, are included. It is noticeable that two of them have diff erent morphologies described as "forma": C. polylepis , now Prymnesium polylepis , "authentic" or "alternate" (Edvardsen and Paasche 1992, Edvardsen et al. 1996, Edvardsen and Medlin 1998; C. palpebralis f. palpebralis or C. palpebralis f. bisquamata (Seoane et al. 2009). As mentioned before, an additional species, referred to as Chrysochromulina sp. 4 (Eikrem and Edvardsen 1999) is now considered as the type species of the new genus Pseudohaptolina (Edvardsen et al. 2011). Th e terminal taxa of the key are 63 because all morphological forms are treated separately. A few of them (freshwater species) are poorly described ( C. inornamenta Wujek & Gardiner, C. chiton var. minuta and C. papillata Gao, Tseng & Guo, C. laurentiana Kling) but still may be identifi ed through this key.

Characters used in the key
Th e key matrix is based on one ecological character (habitat) and 19 morphological descriptors seen in light or electron microscopy, under live conditions or after fi xation for EM observations. Th ey range from cell shape to scale ornamentation. Details of scales can be obtained by specifi c techniques with TEM, such as direct preparations Th omsen 1980, Jensen 1998) and cultures are generally needed. Although rarely used, SEM can also provide interesting results with natural samples (Puigserver et al. 2003). For fragile cells, 3 mL of sample are fi xed with 50 μL of a 1:1 Lugol/Glutaraldehyde (25%) solution, centrifuged on a Th ermanox cell culture coverslip coated with poly-L-lysine (0.1%) for a better adherence of cells, critical point dried and then examined with a fi eld emission scanning electron microscope.

List of descriptors used in the key:
HABITAT: marine, brackish, freshwater SHAPE: spherical-subspherical, elongate to round, lanceolate, saddle-shaped CELL LENGTH, CELL WIDTH: min. and max. sizes are given for each form or species. FLAGELLA: Two fl agella are present and may be equal or sub-equal, in that case the length of the longer and shorter fl agellum are given. HAPTONEMA BEHAVIOR: coiling, rarely coiling, non coiling. HAPTONEMA LENGTH: min. and max. size (in some cases, the haptonema may be very long) NUMBER OF SCALE TYPES: in some cases, scales may be displayed in several layers and show up to four diff erent types but there is always a layer of plate scales as cell covering. SCALE APPENDICES: besides plate scales, a number of diff erent appendices can be observed : spine, cylinder or another typical ornamentation. PLATE SCALE LENGTH AND WIDTH: min. and max. sizes are given for all plate scales.

Software used
Th e interactive key is developed using Xper2 version 2.2 software. It is free software available with multilingual interface and compatible with diff erent OS (Windows, MacOS and Linux) under a creative commons license (BY-CC-ND). You can download it on http://www.infosyslab.fr and fi nd on this website a complete documentation with technical details, user manual and knowledge bases. Xper2 off ers an editor to structure and analyse descriptive data and an interface for interactive free access key (Ung et al. 2010). Keys of various taxonomic groups are already available with Xper2 (Kerner et al. 2011) (Mathieu et al. 2012) (Th omas and De Franceschi 2013).
We installed the interactive key on a web server with Apache2, choosing the English interface. Th is content is under a creative commons license (BY-CC-ND), except when a special information is attached to images.

The knowledge base
Xper2 manages structured descriptive data: all the terminal taxa of the key are described using the same terms (descriptor and character states labels), and so the taxa can be compared automatically. Figure 1 presents the comparison of the two forms attributed to Prymnesium palpebrale (previously Chrysochromulina palpebralis) . Th e diff erent colors allow to point easily where the descriptions are distinct, overlap, or are the same. Here the two forms diff er on scale type number and appendices.
In the same way the comparison of the fi ve species ( Haptolina brevifi la , H. ericina , H. fragaria , H. herdlensis and H. hirta ) previously known as Chrysochromulina but attributed in 2011 in the new genus Haptolina (Edvardsen et al. 2011) shows that these species share few attributes used in the key (Figure 2).

The online interactive key
Our key of the Chrysochromulina species is a free access key accessible at http://www. obs-banyuls.fr/chrysochromulina. It off ers an interactive and fl exible way to identify these phytoplanktonic species. A classical polytomous key consists of a series of questions (characters), each one off ering alternative statements (Hagedorn et al. 2010). A free access key is a more fl exible identifi cation key: the sequence of choices is defi ned by the user preventing these of characters diffi cult or impossible to observe. Figure 3 shows the screen during an identifi cation process. Each item (terminal taxon of the key) is documented by a text including nomenclatural data, type locality, literature references and morphological data, and is illustrated by several images. Descriptors and character states are also documented and illustrated.
At each step, the user may ask the software to fi nd the best characters to distinguish the possible taxa. Th ree diff erent measures are proposed Xper, Jaccard, and Sokal & Michener (as "Best descriptors" in the select box). For each pair of remaining taxa, each coeffi cient measurement compares the possible states and the fi nal result is the sum for all the pairs. Xper coeffi cient checks only if there is no overlap (it means the two taxa may be completely distinct on this character) and so the measure for one pair of taxa is 0 (if overlapping) and 1 (if no overlapping). Jaccard coeffi cient was initially developed to compare sets of binary characters; here the states are considered as the binary characters and the

Conclusions
Identifi cation of Chrysochromulina species has long been reserved to specialists as it is a major diffi culty for most phytoplanktonologists. Th e cells are very small, often overlooked or placed as "unidentifi ed" species in fi eld studies. Cultures and specifi c preparations are generally needed to get relevant information on morphological features. A key for identifi cation of Scandinavian species ), based on TEM observations of cultures has only been published in PhD Th eses (Jensen1998 and Eikrem 1999). A list of species as part of toxic haptophytes was published by Moestrup & Th omsen in a manual on harmful marine microalgae (Moestrup and Th omsen 2004). However original descriptions are not always available for researchers. In this interactive key, all species found in the literature are treated and information necessary for their identifi cation is provided. Th is key is a very powerful tool for a taxonomic work on the genus and is therefore strongly recommended, especially for phytoplanktonologists working on nanofl agellates. Th e content of the key was carefully checked and tested with information on species characteristics found in the Scandinavian key , Eikrem 1999 and unpublished data obtained by one author (M-J C-D). Among all descriptors used in this key, those concerning the scale description are most important and the mean number of required characters to identify a species is 4.3. SEM preparations from fi eld samples seem promising for identifi cation of these species (Puigserver et al. 2003 and unpublished illustrations shown in the key). Th e choice of characters introduced without order is also an important advantage as compared to a classical key: characteristics of a typical scale may be enough for a specifi c identifi cation.