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Cryptomonad taxonomy in the 21st century:
The fi rst two hundred years
GIANFRANCO NOVARINO
Abstract. Nearly two centuries have gone by since Ehrenberg made the fi rst known observations on
cryptomonads, a group of bifl agellate unicellular microalgae/protists/chromists found in all aquatic habi-
tats, often with very high population densities. Cryptomonad identifi cation using light microscopy has
proved diffi cult ever since Ehrenberg’s time and it is with these premises that cryptomonad taxonomy
took form until the mid-20th Century. Later, new ultrastructural information revolutionised traditional
cryptomonad taxonomy and systematics to the point that the two most recent formal classifi cation sys-
tems of cryptomonads are based on the cell ultrastructure. Beginning in the 1990’s an ever-increasing
amount of molecular genetic information has also become available, focussing either on the phylogeny
of the cryptomonads as a whole or the phylogenetic relationships between the various cryptomonad
supra-generic groupings, genera and, to a lesser extent, species. Crucial to the study of cryptomonad
identifi cation, taxonomy, systematics and phylogeny is the concept of what constitutes a cryptomonad
“species”. The simplest one is the nomispecies concept, whereby a taxon at or below the species level
is deemed to have a real existence for the simple reason that it has been described and given a Linnean
binomen (whether valid or not) in the literature. The cryptomonad nomispecies concept is entirely or
almost entirely a morphological one. Presently, 21 genera are recognized with not less than 429 nomispe-
cies in total, a fi gure more than twice the commonly cited one for the cryptomonads (Cryptomonas, 200;
Chroomonas sensu auctorum, 80; “Rhodomonas”, 40; Chilomonas, 30; Hemiselmis, 20; Plagioselmis,
Hillea, Pyrenomonas and Rhinomonas, 10 each; Goniomonas, Proteomonas and Teleaulax, 3 each;
Chroomonas sensu stricto 2-?; Cryptochloris, Campylomonas, Falcomonas, Hanusia, Geminigera,
Guillardia, Komma and Storeatula, 1 each). The process of estimating the number of cryptomonads
still to be described is hampered by the existence of several alternative species concepts which apply
equally to the group. Based on a fi gure of 429 nomispecies, a revised estimate of some 780 existing
(morpho)species is suggested here, or about 550 when a synonymy rate of 30% is allowed for. The
future development of the cryptomonad species concept and the taxonomy on which it is based will
have to take into account, and successfully respond to, challenges presented by the study of the cryp-
tomonad life-cycle, ecology, and molecular genetics. The present and future impact of these challenges
is discussed, and a possible perspective for future cryptomonad research is presented.
Key words: Cryptomonadea, cryptomonads, Cryptophyceae, ecology, morphological variability, number
of species, phylogeny, species concepts, systematics, taxonomy, ultrastructure
Gianfranco Novarino, Private Laboratory, 93 Sinclair Road, London W14 0NP, U.K.; and Dept. of
Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, U.K.; e-mail: gianfranco@
cryptomonads.net and gn@nhm.ac.uk
Phycological Reports: Current advances in algal taxonomy and its applications: phy-
logenetic, ecological and applied perspective. Institute of Botany Polish Academy of
Sciences, Kraków, 2012: 19-52.
2 Phycological Reports: Current advances in algal taxonomy
Introduction
This paper summarises the state of cryptomonad taxonomy and analyses the impact
of three challenges presented to it (especially in connection with the crucial concept
of what constitutes a cryptomonad “species”) by advances in the study of the cryp-
tomonad life-cycle, ecology, and molecular genetics. The bias placed on each one
of these aspects refl ects the author’s own background. A comprehensive account of
cryptomonad morphology, ultrastructure, biology, ecology and phylogeny is outside
the immediate scope of this paper and the reader is referred to existing reviews (No-
varino 2003 and references therein), although relevant information will be mentioned
where necessary.
Cryptomonads are unicellular, bifl agellate, mostly photosynthetic microalgae/protists.
(N.B. These terms are used here in a descriptive sense with no implied phylogenetic
meaning; other terms – e.g. chromists – could equally be used.) They hardly escape the
attention of phytoplankton investigators owing to their widespread occurrence in virtu-
ally all aquatic habitats (often in high or very high numbers), the peculiar cell colours
(roughly speaking, either “red” or “blue-green” owing to the presence of phycobilins
– either a phycoerythrin or a phycocyanin in any given cell – as principal accessory
photosynthetic pigments: Hill & Rowan 1989; Novarino 2003), and their characteristic
swimming behaviour, which has been described as “recoiling” (Novarino 2003). There-
fore, it is not too much of a surprise that Christian Gottfried Ehrenberg (1795–1876),
the widely acknowledged fi rst discoverer of many different kinds of microorganisms,
observed cryptomonads as early as in the second or third decade of the 19th Century.
The fi rst mention of cryptomonads (Ehrenberg 1831a) appears to be in the Symbolae
Physicae seu Icones et Descriptiones Animalium Evertebratorum sepositis Insectis, a rare
series of synoptic tables on invertebrate animals in which Ehrenberg fi rst introduced the
genus names Chilomonas and Cryptomonas without accompanying verbal descriptions
or drawings. At around the same time he also mentioned those two genera in another
publication (Ehrenberg 1831b, printed in 1832), but Latin and French diagnoses, ex-
tensive descriptions and informative drawings were only provided on a later occasion
(Ehrenberg 1838). Owing to the much higher information content and quality provided
there – as opposed to little or none in 1831–32 – the starting date for cryptomonad
taxonomy adopted here is that of 1838 although this is in disagreement with the Index
Nominum Genericorum Plantarum (Farr & Zijlstra 1996+).
Ehrenberg described several morphological details in the cells he observed, e.g. cell
colour, shape, size, fl agella and various inclusions, a remarkable achievement for his day
and age (Fig. 1). Although his “complete-organism” theory is notorious for not neces-
sarily leading him to interpret correctly everything he saw (be it in the cryptomonads or
other microorganisms), Ehrenberg’s relentless observational approach laid the founda-
tions for the whole of cryptomonad taxonomy for more than a century to follow. The
characters he observed were invariably used in many future taxonomic descriptions, in
addition to others as and when they were discovered and correctly interpreted – e.g.
pyrenoids, trichocysts (also called ejectosomes = ejectisomes), starch granules, eyespots
etc.. Notable taxonomists active during the century that followed Ehrenberg included
G. Novarino: Cryptomonad taxonomy in the 21st century 3
Adolf Pascher (1881–1945), Heinrichs Skuja (1892–1972) and Roger William Butcher
(1897–1971).
Adolf Pascher’s fi ne microscopical observations (e.g. Pascher 1911, 1913) enabled
him to provide detailed and reliable taxonomic descriptions accompanied by accu-
rate, informative drawings (Fig. 2). Although he prolifi cally described many new taxa
within other groups of microalgae and protists, his contribution to the total number of
described cryptomonad taxa has been relatively small. However, several of his taxo-
nomic descriptions have proved long-lasting and some of his taxa are commonly cited
as regular constituents of the freshwater cryptomonad fl ora. Pascher also attempted to
lay the foundations of modern cryptomonad classifi cation by subdividing the crypto-
monads into permanently motile ones and periodically non-motile ones, a view which
has nonetheless fallen into disuse.
Heinrichs Skuja (Skuja 1939, 1948, 1956) also provided fi ne microscopical and
taxonomic observations, and the quality of his drawings is perhaps the highest ever
Fig. 1. Ehrenberg’s original illustrations of Cryptomonas ovata Ehrenb. (left) and C. erosa Ehrenb. (right). From
Ehrenberg (1838).
Fig. 2. Pascher’s original illustrations of Protochrysis phaeophycearum Pascher. Very likely the genus Protochrysis
is the same as Hemiselmis Parke on morphological grounds (see Novarino 2003). From Pascher (1911).
4 Phycological Reports: Current advances in algal taxonomy
attained within the cryptomonads (Fig. 3). He described a larger number of cryptomonad
taxa than Pascher, and thanks to his very reliable and detailed taxonomic descriptions
and drawings these taxa are still recorded frequently by freshwater phycologists. His
contribution to the alpha-taxonomy of this group was very signifi cant, as shown by
the fact that his seminal works (Skuja 1939, 1948, 1956) are still used as fundamental
identifi cation aids by many workers all around the world.
Roger William Butcher, a botanist interested both in higher plants and microscopic
algae, emerged in the 1950’s-1960’s as a prolifi c producer of taxonomic descriptions
of marine and brackish cryptomonads. In a monograph of British marine cryptomonads
(Butcher 1967) he attempted to provide an identifi cation guide to the taxa found in his
newly established laboratory cultures. Being also interested in higher-level classifi cation
he introduced a new cryptomonad classifi cation system based on the morphology of the
Fig. 3. Skuja’s illustrations of Cryptomonas ovata Ehrenb. Note the regular arrangement of hexagonal elements,
which may correspond to the periplast plates (see Novarino 1993). From Skuja (1939).
Fig. 4. Absorbance spectra of phycoerythrin extracts from Proteomonas pseudobaltica (Butcher) Novarino and
Campylomonas sp. The absorbance peaks (at 545 and 560 nm, respectively), identify the P. pseudobaltica pigment
as Cr-PE I and the Campylomonas pigment as Cr-PEIII.
G. Novarino: Cryptomonad taxonomy in the 21st century 5
anterior cell region from which the fl agella arise (the “furrow-gullet system”), which ap-
proach was in marked contrast with his choice of primary identifi cation characters at the
species level, i.e. the cell colour (Butcher 1967). His rich drawings of cryptomonad cells
and their phenotypic variability, encompassing a wide range of subtle colour variations
(verbally described with the aid of an offi cial British guide to colours for painters and
decorators), are now known to show cells in which the varying contents of water-soluble
phycobilin pigments have produced variations in the visible cell colour by means of
a greater or lesser masking effect over the other accessory pigments and the chlorophylls.
Phycobilins are photosynthetic pigments composed of an open tetrapyrrole chain plus
a protein moiety, and their absorbance spectra differ characteristically between the vari-
ous types (Fig. 4). Nowadays, as a result of detailed information on phycobilin spectral
features there is a resurgence of interest in cell “colour” – i.e., phycobilin identity – and
its signifi cance within the context of cryptomonad identifi cation, taxonomy, classifi ca-
tion and phylogeny (Table 1).
Diffi culties in cryptomonad identifi cation and taxonomy
Unfortunately Pascher, Skuja and Butcher were exceptions rather than the general rule.
Many formal taxonomic protologues from the time of Ehrenberg to the mid-20th Century
were little more than skeletal verbal descriptions accompanied by minimalist drawings
(Fig. 5). Historically, descriptions of this sort have been one of the main diffi culties
hindering cryptomonad identifi cation and taxonomy. Other diffi culties – somewhat arbi-
trarily subdivided in “anthropic” (= cultural = subjective) and “non-anthropic” (= natural
= objective) – are summarised below.
Anthropic diffi culties. Anthropic diffi culties are just that: diffi culties caused by humans.
As such they can be easily solved, at least in theory, as long as there is a commitment to
doing so. The most basic diffi culties are those posed by inadequate or non-existing iden-
tifi cation tools (unavailability of up-to-date monographic revisions of the cryptomonads,
updated taxonomic keys or automatic identifi cation aids; diffi cult or impossible access
to the abundant taxonomic literature necessary for identifi cation, including many of the
older, hard to fi nd publications), and inadequate observation methodology (substandard
Table 1. Taxonomic distribution of Cr-phycobilins in selected cryptomonad genera. PC = phycocyanin, PE =
phycoerythrin. Based on Hill and Rowan (1989), see also Novarino (2003). Generally speaking, the absorbance
spectra of phycoerythrin pigments have one peak only while phycocyanins have two.
Phycobilin Major peak (nm) Minor peak (nm) Genera
Cr – PE I 540–550 – Geminigera, Plagioselmis, Proteomonas, Pyrenomonas/
Rhodomonas emend., Rhinomonas, Storeatula, Teleaulax
Cr – PE II 555 – Hemiselmis subgen. Hemiselmis
Cr – PE III 560–566 – Campylomonas, Cryptomonas sensu lato, Cryptomonas
sensu stricto
Cr – PC 569 569 630 Falcomonas
Cr – PC 615 615 577–585 Hemiselmis subgen. Plagiomonas
Cr – PC 630 625–630 580–584 Few species of Chroomonas (sensu auctorum)
Cr – PC 645 640–650 580–585 Most species of Chroomonas (sensu auctorum)
6 Phycological Reports: Current advances in algal taxonomy
or inadequately set-up microscopes, inadequate fi xation protocols, inadequate observation
techniques). The fi rst set of diffi culties can be addressed by making all the information
which is necessary for taxonomic identifi cation available on a centralised basis, a requisite
which is served well by an Internet-based facility. One example is Novarino and Gaddini
(2010), an independent website under construction at the time of writing which is aimed
at collating as much information on cryptomonads as possible, especially in connection
with taxonomic identifi cation. Another approach is that of integrating as much taxonomic
data as possible and making it available to non-specialists in a digested form which
includes polytomous keys and multi-lateral cross-referencing of information (Novarino
2003). As far as inadequate methodology is concerned, it has long become apparent
that light microscopy (LM) on its own is usually insuffi cient for identifying all known
cryptomonads in detail, and it is precisely this realisation that has contributed over the
years to the “ultrastructural revolution” of the mid-late 20th Century, i.e. the discovery
of new ultrastructural features peculiar to the cryptomonads and their subsequent use
in taxonomy, as will be mentioned later.
Other anthropic diffi culties include the description of monospecifi c genera, which
has been seen as undesirable (even though it may be absolutely justifi ed and unavoid-
able) because it may lead to an incorrect appreciation of interspecifi c variability within
those genera. This is detrimental if new species are found eventually within these genera
because an emendation of the generic diagnoses becomes necessary at that stage (No-
varino 2003). Typifi cation – the process of fi xing nomenclatural types for the names of
all described taxa, and subsequently using them in the most fruitful way possible for
identifi cation, taxonomic and systematic purposes – has always caused diffi culties. Within
the cryptomonads there has been an historical disregard for the type method, which is
very unfortunate because it is the fundamental principle of the Codes of Nomenclature
which apply to the cryptomonads, leading to nomenclatural stability in the long run
(Silva 1996; see also Novarino 2003).
Fig. 5. A minimalist type fi gure: Conrad and Kufferath’s original illustration of Chroomonas daucoides Conrad
et Kufferath reproduced in black and white. The original drawing shows the chloroplasts in light blue colour. This
species has been recombined as Falcomonas daucoides (Conrad et Kufferath) Hill, the type and only known species
of the genus Falcomonas. From Conrad and Kufferath (1954).
G. Novarino: Cryptomonad taxonomy in the 21st century 7
The disregard for typifi cation within the cryptomonads is deemed here to derive
largely from the widespread but incorrect belief that preserved type materials of cryp-
tomonads do not exist. On the contrary, original type materials may well exist and it
may be possible to re-examine them ultrastructurally, even if they are over 100 years
old. This is shown by the case of the preserved type specimens observed by Hansgirg
(1885) during the description of the genus Chroomonas, which have been examined
using scanning electron microscopy (SEM) (Novarino 2003). Such a case may have
important consequences for establishing the true ultrastructural identity of the genus
because the current concept of Chroomonas does not fi t with Hansgirg’s own type
specimens. Chroomonas sensu auctorum has rectangular periplast plates, whereas the
Chroomonas type specimens – i.e. those examined by Hansgirg himself – have hexagonal
plates (Novarino 2003).
Other diffi culties include the long-standing gap between ecological studies on the
one hand and cryptomonad taxonomy and systematics on the other; and the state of fl ux
of cryptomonad classifi cation systems, mostly in relation to the availability of an ever-
increasing quantity of molecular phylogenetic data. Both of these will be discussed sepa-
rately below, as they pose unique challenges to the whole of cryptomonad taxonomy.
Finally, from a philosophical point of view it has been argued (Novarino 1990, 2003)
that authoritative viewpoints expressed by eminent specialists can exercise a negative infl u-
ence on the taxonomists’ morale and confi dence, and consequently on the future develop-
ment of taxonomy as a whole. In the case of the cryptomonads, the view expressed by
Pringsheim (1968) whereby cryptomonads are simply “impossible to classify” seems to have
persisted for a long time, to the detriment of the advance of cryptomonad taxonomy.
Non-anthropic diffi culties. Non-anthropic diffi culties are more complex to address
owing to the very fact that they are inherent in the organisms under study. As mentioned,
the notorious scarcity of characters readily visible with the LM in the cryptomonads
is one of the main contributory factors which has led to the ultrastructural revolution
mentioned below. Additionally, the high degree of phenotypic variability shown by
cryptomonad cells may be partly environmentally-dependent (i.e. related to abiotic
factors and/or ecological interactions between co-existing or competing populations),
and as such is discussed later within the challenge posed to cryptomonad taxonomy by
ecology. Finally, the observed co-existence of contrasting morphotypes within a single
species (the “crypto-campylomorph” question), constitutes a third important challenge
to cryptomonad taxonomy and will also be discussed separately below.
The ultrastructural revolution
Beginning in the mid-1960’s electron microscopy (EM) has revealed a multitude of previ-
ously unknown cellular features, many of which have been included in a comprehensive
review of taxonomically valuable characters within the cryptomonads (Klaveness 1985).
Since then, several ultrastructural characters have been used for describing new genera and
species, and amending existing generic diagnoses (Hill & Wetherbee 1986, 1988, 1989,
1990; Hill 1991a; 1991b; Novarino 1991a; 1991b; Novarino & Lucas 1993a; Novarino
et al. 1994; Kugrens et al. 1999). Subsequently, two attempts have been made to erect
8 Phycological Reports: Current advances in algal taxonomy
formal classifi cation systems of the cryptomonads based on ultrastructural characters
(Novarino & Lucas 1993a, 1995; Clay et al. 1999).
Paradoxically the ultrastructural revolution also brought about a new diffi culty,
i.e. that of relating classical taxa (long-described ones whose descriptions are based
on LM only) to newer ultrastructural descriptions, as exemplifi ed by the question of
Rhodomonas, a genus originally described by Karsten (1898) and then emended nearly
a century later based mainly on electron microscopy (Hill & Wetherbee 1989). Because
the examined strains (Hill & Wetherbee 1989) showed the characteristic ultrastructural
features of the genus Pyrenomonas Santore (1984), it was argued (Hill & Wetherbee
1989) that Pyrenomonas was a later (junior) synonym of Rhodomonas and therefore it
was to be abandoned. Elsewhere it was strongly contended that the name Rhodomonas
emend. Hill & Wetherbee (1989) was the later synonym and therefore it was to be
abandoned in favour of Pyrenomonas, which name ought to have been adopted by Hill
& Wetherbee (1989) rather than Rhodomonas (Novarino 1991a; Novarino & Lucas
1993b). In essence, the name Rhodomonas emend. Hill et Wetherbee (1989) is a later
unnecessary name for Pyrenomonas Santore (1984), i.e. a nomen superfl uum (Novarino
& Lucas 1993b). There is no evidence whatsoever that the genus Rhodomonas in the
original sense of Karsten (1898) has the ultrastructural identity of the cryptomonads
examined by Hill & Wetherbee (1989), or of any other cryptomonads which have been
examined ultrastructurally, simply because the ultrastructure of the specimens examined
by Karsten (1898) is totally unknown, and will always be unless Karsten’s original type
specimens will be rediscovered and examined ultrastructurally.
A selection of ultrastructural features which are consistently accorded taxonomic
signifi cance at various levels is summarised in Table 2. The use of characters revealed by
SEM (Figs 6–9) is particularly advantageous owing to the smaller technical diffi culties
involved with specimen preparation and examination compared to transmission electron
microscopy (TEM). This has led to SEM being used as a routine identifi cation tool
during ecological surveys (Novarino et al. 1997; Bérard-Therriault et al. 1999; Barlow
& Kugrens 2002; Novarino 2005; Cerino & Zingone 2006; Xing et al. 2008). However,
in spite of the great taxonomic usefulness of SEM, not all cryptomonads can be identifi ed
conclusively using this observation method alone because further information might be
necessary on the identity of the accessory photosynthetic phycobilin pigment, the detailed
architecture of the cell covering (periplast, as revealed by freeze-fracture/etch methods)
and the internal cell ultrastructure, especially the position of the nucleomorph (as revealed
by transmission electron microscopy). The possible occurrence of preparation artefacts
should also be borne in mind (discussed at length in Novarino 2005).
Kind of periplast. Cryptomonads possess a typical cell covering termed the periplast.
There is evidence to suggest that the periplast was detected during two early LM inves-
tigations (Novarino 1993). However, such cases are exceptional and only EM has made
it possible to elucidate the periplast structure in detail. Since the fi rst EM observations
(Lucas 1970; Gantt 1971; Hibberd et al. 1971; Faust 1974), our knowledge of periplast
architecture has progressed considerably thanks especially to freeze-fracture studies
but also SEM. Periplast construction is very diverse but there is always a protein layer
underneath the cell membrane (internal periplast component, IPC). The IPC can be made
G. Novarino: Cryptomonad taxonomy in the 21st century 9
up either of a single element (i.e. it is a continuous sheet), or a series of discrete plates.
There is also an external periplast component (EPC), which is very variable in structure,
being composed for instance of variously conformed fi brils and/or small (150–200 nm
but sometimes down to 80 nm) organic scales. Some cryptomonads with a plated IPC
have an EPC composed of plates similar to the IPC. One exception is Guillardia, which
has a sheet-like IPC and an EPC composed of large plates (Hill & Wetherbee 1990).
Plated versus non-plated periplasts are used as a taxonomically signifi cant character at
the generic level (Santore 1984; Kugrens & Lee 1987; Hill & Wetherbee 1988, 1989,
1990; Hill 1991a, 1991b; Novarino 1991a, 1991b; Novarino & Lucas 1993a, 1993b;
Novarino et al. 1994; Novarino & Lucas 1995; Clay et al. 1999; Kugrens et al. 1999).
The SEM is usually capable of revealing the sheet-like appearance of the IPC in taxa
with a non-plated IPC, and the plated appearance of the IPC or the EPC in those taxa
with a plated IPC and/or EPC. Among the possible preparation artefacts affecting the
appearance of the periplast in the SEM, cell shrinkage is the one which is most likely
to occur; another possible artefact is given by the discharge of the ejectosomes present
at the cell periphery (Novarino 2003, 2005). Freeze-fracture studies (Brett & Wetherbee
1986; Wetherbee et al. 1986; Kugrens & Lee 1987; Wetherbee et al. 1987) have pro-
vided more detailed insight into the intimate architecture of the cell surface, showing
that cryptomonad periplasts are highly diversifi ed and at the same time providing more
general information on the eukaryotic cell surface (Brett et al. 1994). As a consequence
there has been an increasing use of freeze-fracture investigations in purely taxonomic
studies (e.g. Hill & Wetherbee 1986, 1988, 1989, 1990; Hill 1991a, 1991b; Clay & Ku-
grens 1999), but unfortunately these observations are diffi cult to carry out during routine
taxonomic surveys.
Figs 6–9. Examples of cryptomonads as seen in SEM. 6 – Plagioselmis prolonga Butcher ex Novarino, Lucas et
Morral, 7 – Proteomonas pseudobaltica (Butcher) Novarino, 8 – Teleaulax amphioxeia (Conrad) Hill, 9 – Rhinomonas
reticulata (Lucas) Novarino. Plagioselmis and Rhinomonas (plated periplast) are representative of “cryptomorph”
morphology while Proteomonas and Teleaulax are representative of “campylomorph” morphology. Scale bars
= 2 μm (Fig. 6), 5 μm (Figs 7, 8) or 2.5 μm (Fig. 9).
10 Phycological Reports: Current advances in algal taxonomy
Table 2. Taxonomic distribution by genera of selected ultrastructural features useful for identifi cation. E: eyespot; EPC: external periplast component; F: fl agella; Fu: furrow;
IPC: internal periplast component; MVB: mid-ventral band; NM: nucleomorph; P: periplast; PP: periplast plates; PR: periplast raphe.
Genus
P: IPC non-plated, P
sheet-like in SEM
P: IPC and/or EPC
plated, P plated in
SEM
PP rectangular
PP distinctly
hexagonal
PP rectangular-
hexagonal (square
with bevelled edges)
PR sometimes
observed in SEM
Non-artefactual Fu
sometimes observed
in SEM
MVB sometimes
observed in SEM
F ornamentation not
typical “2+1” pattern
NM
E some-
times
present
Campylomonas √ √ √ extrapyrenoidal
Chilomonas √ √ √ extrapyrenoidal
Chroomonas sensu auctorum √ √ √ extrapyrenoidal √
Chroomonas sensu strictu √ √ extrapyrenoidal ?
Cryptochloris √√?
Cryptomonas (partim) √ √ √ √ extrapyrenoidal √
Falcomonas √ √ √ √ extrapyrenoidal
Geminigera √extrapyrenoidal
Goniomonas √ √ √ absent
Guillardia √ √ extrapyrenoidal
Hanusia √extrapyrenoidal
Hemiselmis √ √ √ extrapyrenoidal
Hillea √ ?
Komma √ √ extrapyrenoidal
Plagioselmis √ (except on the
cell tail, where
it is non-plated)
√ √ √ extrapyrenoidal
Proteomonas (diplomorph stage) √ √ extrapyrenoidal
Proteomonas (haplomorph stage) √ √ √ extrapyrenoidal
Pyrenomonas/
Rhodomonasemend.
√ √ √ √ intrapyrenoidal
Rhinomonas √ √ intrapyrenoidal
Storeatula √intrapyrenoidal
Teleaulax √ √ extrapyrenoidal
G. Novarino: Cryptomonad taxonomy in the 21st century 11
Shape and size of the periplast plates when plates are present. When plated periplasts
are present, the size of the plates can be used to delimit species within particular genera
(Meyer 1984; Novarino 1991a, 1991b; Novarino & Lucas 1993a; Novarino et al. 1994;
Kristiansen & Kristiansen 1999). When measuring the periplast plates, the possible oc-
currence of shrinkage artefacts should also be borne in mind and it may require some
interpretation on the part of the examiner (Novarino 2005).
Presence or absence of a posterior tail. ‘Tails’ – i.e. acute posterior ends which are
often curved ventrally or dorsally – can be observed also with the LM, but the SEM
can show whether or not the periplast is of the same kind present on the rest of the cell
surface. This feature is taxonomically signifi cant at the generic level because it is diag-
nostic of Plagioselmis, which has a periplast composed of hexagonal plates on the main
portion of the cell body and a non-plated periplast on the tail (Novarino et al. 1994).
Presence or absence of a mid-ventral band in the region between the posterior end
of the furrow and the posterior end of the cell. Mid-ventral bands are present in a few
genera. They are easily observed with the SEM, where they appear as a cord-like structure
on the ventral cell surface in the posterior region of the cell. Within individual genera
they do not appear to be present in all of the species (Hill & Wetherbee 1989; Hill 1991b;
Novarino et al.1994), suggesting that their taxonomic value is at the species level.
Presence or absence of a periplast raphe. The raphe – a line at the posterior end of
the cell where the periplast plates seem to converge – is a unique feature of some species
of the genus Chroomonas sensu auctorum. It may be visible using SEM, although freeze-
fracture methods for TEM are preferable (see Novarino 2003 and references therein).
Scales. Cryptomonads may possess characteristic organic scales over the cell body
and/or the fl agella. (e.g. Santore 1983; Lee & Kugrens 1986). The typical size of the
body scales is ca. 150–200 nm, although scales down to about 80 nm in size have been
found in sectioned material of Plagioselmis (Novarino et al. 1994). Freeze-fracture
investigations often reveal more detail than wholemounts. The taxonomic distribution
and signifi cance of cryptomonad scales is not entirely clear at present and therefore it
requires further investigation.
Morphology of the vestibular region of the cell from which the fl agella arise, espe-
cially the presence or absence of a true, non artefactual ventral furrow. This structure
has been the object of much controversy ever since the early LM taxonomic descriptions.
The term ‘furrow’ refers to a shallow groove on the ventral face of the cell, of variable
length and width. Some of the early light microscopists described cryptomonads with
open furrows, but the exact architecture of these structures (especially their spatial rela-
tionships with other vestibular structures such as the closed, tubular gullet) was virtually
impossible to establish with certainty using light microscopy only. Since the advent of
electron microscopy, SEM has been the tool of choice for studying these structures. It
has been hypothesized (Santore 1984) that furrows are always preparation artefacts, and
therefore they have no taxonomic value at all, or that they are never artefactual (Kugrens
et al. 1986; Hill & Wetherbee 1989) and are taxonomically signifi cant at the generic level
(Clay et al. 1999). A more complex view is that the term ‘furrow’ may encompass arte-
factual and non-artefactual structures alike (Novarino 1991b), and it is possible to judge
whether or not an observed furrow is an artefact based on a few simple considerations
12 Phycological Reports: Current advances in algal taxonomy
(presence or absence of obvious signs of shrinkage or collapse in other regions of the
cell; frequency of the occurrence of observed furrows in a sample of cells – say 30–40
– belonging to the same species; and, in cryptomonads with a plated periplast, presence
or absence of periplast plates on the internal face of the furrow, because true furrows are
never lined internally with discrete periplast plates of the same kind as those found on
the rest of the cell). It has been argued that true furrows are taxonomically signifi cant
at the species rather than the genus level because they are not necessarily present in
all of the species of a particular genus (Novarino 1991b; Novarino et al. 1994). The
function of the furrow/gullet system of cryptomonads is still unknown. Cryptomonads
with a closed tubular gullet may occasionally harbour bacteria in the gullet itself, e.g.
spirochaete bacteria in Rhinomonas (Novarino 2003).
Arrangement, absolute and relative length of the fl agella. Flagella can be observed
also with the LM, but their length and point of insertion can be determined much more
accurately with the SEM. Features such as the median/subapical versus apical insertion
point of the fl agella and their absolute and relative length have been considered to be
taxonomically signifi cant since the times of the early LM descriptions (at the genus and
species level, respectively). This view has been upheld also in more recent ultrastructural
and taxonomic investigations (Hill & Wetherbee 1988; Novarino & Lucas 1993b; Clay
& Kugrens 1999a).
Arrangement of fl agellar appendages. The nature and arrangement of fl agellar ap-
pendages in cryptomonads are quite diverse (Morrall 1980; Kugrens et al. 1987). The
taxonomic signifi cance of these appendages is not entirely clear but some genera al-
ways show a characteristic pattern. The typical fl agellar appendage of cryptomonads is
a tubular hair (mastigoneme) with terminal fi lament(s). Whole mounts for TEM are the
choice observation method but the SEM may still show the appendages in adequately
fi xed cells, revealing whether or not they are arranged according to the typical pattern
(see Morrall 1980; Kugrens et al. 1987) which is two rows on the dorsal (usually longer)
fl agellum and one row on the ventral (usually shorter) fl agellum; this is referred to here
as the ‘2+1’ pattern. However, there is considerable variation (Morrall 1980; Kugrens
et al. 1987). Campylomonas and Chilomonas both have a single row of mastigonemes
on each fl agellum (Kugrens et al. 1987; Gromov et al. 1998). Goniomonas has been
reported as having a similar arrangement of mastigonemes (Morrall 1980), or peculiar,
non-tubular ‘spike’ appendages on one fl agellum only (Kugrens et al. 1987; see also
Novarino 2003).
Presence and position of the nucleomorph. The cryptomonad nucleomorph, a distinct
cell organelle resembling an eukaryotic nucleus (Greenwood 1974) and now known to
represent the vestigial nucleus of a red algal-like photosynthetic endosymbiont from
which the present-day cryptomonad chloroplast has evolved (e.g. Hansmann et al. 1985;
Ludwig & Gibbs 1985; Hansmann et al. 1986), is located in the periplastidial compart-
ment and within a particular genus it occurs in one of two basic positions, i.e. embedded
in a groove within the pyrenoid (‘intrapyrenoidal nucleomorph’) or outside the pyrenoid
(‘extrapyrenoidal’). The intra- versus extrapyrenoidal position was fi rst considered to
be systematically signifi cant at the order level elsewhere (Novarino & Lucas 1993b)
and later expanded upon and incorporated into a newer classifi cation system (Clay et
G. Novarino: Cryptomonad taxonomy in the 21st century 13
al. 1999). It is sometimes possible to locate the nucleomorph by fl uorescence LM if the
cells have been stained with the DNA-specifi c fl uorochrome DAPI (Ludwig & Gibbs
1985). There is also some evidence suggesting that in the 1950s the nucleomorph may
have been observed unknowingly using conventional histochemical staining (Novarino
1993). However, TEM is the choice method. Goniomonas is the only known crypto-
monad genus where a nucleomorph is lacking (Morrall 1980; Kugrens & Lee 1991;
Martin-Cereceda et al. 2010), and as such it is believed to be closely related to the
ancestral cryptomonad cell which acquired a photosynthetic endosymbiont and led to
the evolution of the group. This view is supported by molecular evidence (McFadden et
al. 1994). The three known genera with an intrapyrenoidal nucleomorph (Pyrenomonas/
Rhodomonas emend., Rhinomonas and Storeatula) are all grouped in a single order (No-
varino & Lucas 1993b; Clay et al. 1999), whose separation is supported by molecular
studies (Cavalier-Smith et al. 1996; Hoef-Emden et al. 2002; Deane et al. 2002). In all
other genera in which the presence and position of a nucleomorph has been established
the nucleomorph is extrapyrenoidal.
Occurrence of a true eyespot. Several species of Chroomonas sensu lato may be
seen to possess putative eyespots in the LM. TEM can confi rm or refute whether these
are true eyespots rather than other structures. True eyespots appear as a stalked exten-
sion of the pyrenoid containing several electron dense globules at the periphery, i.e. in
the direction facing the cell surface (Lucas 1982).
The challenge of alternating morphologies
For a given character (be it morphological or otherwise) to be signifi cant at a given
taxonomic level, its variability within the taxon under consideration (“infra” variability)
must be smaller than the variability between that taxon and similar albeit distinct taxa
(“inter” variability), i.e. a character showing more inter- than infra- variability is more
signifi cant than one for which the opposite is true (Pankhurst 1978). In the particular
case of cryptomonad species defi ned morphologically, the search for taxonomically
signifi cant morphological characters requires therefore a preliminary assessment of their
inter- and infraspecifi c variability.
Unfortunately however, in spite of their universally recognised high level of variabil-
ity, the cryptomonads have been the object of only few formal studies on morphological
variability as it occurs in nature (Lund 1962; Javornicky 1976; Willen et al. 1980). It
could well be postulated, therefore, that at least some of the described species are simple
morphological variants of other, highly variable species, but unfortunately this is diffi cult
to demonstrate in practice. Even if culture studies were to reveal the existence of certain
morphological variants in a given species, it would be by no means implicit – let alone
demonstrated – that the species under examination effectively exhibits in nature the entire
spectrum of variability seen in culture. For instance, certain deviations from “normal”
morphology might appear only in the presence of extreme culture conditions, which might
rarely (or never?) occur in nature. Examples are offered by the electron-dense bodies
consistently present under the periplast of Chroomonas grown at very high irradiance,
which are invariably absent under normal irradiance conditions and have not been seen
14 Phycological Reports: Current advances in algal taxonomy
to occur in nature (Novarino & Lucas 1993a); and the frequent presence of various kinds
of non-motile cells in ageing cultures of many cryptomonad species (Pringsheim 1944),
which are not necessarily found in nature unless the specifi c environmental conditions
required present themselves (e.g. Javornický & Hindák 1970).
As mentioned, cryptomonad periplast features have been used extensively as taxo-
nomic characters at the genus and species levels. A recent challenge to this standpoint
comes from the existence of three species (Table 3), in each of which cells with pro-
nounced morphological (ultrastructural) differences co-exist and are believed to alternate
regularly within the life cycle (Hill & Wetherbee 1986; Hoef-Emden & Melkonian 2003).
This is consistent with the fi nding that a number of morph pairs have identical sequences
of certain genes (Hoef-Emden & Melkonian 2003) or have been interpreted as consisting
of a haploid and a diploid cellular stage (Hill & Wetherbee 1986). “Cryptomorph” cells
have a plated periplast (e.g. Fig. 9), whereas “campylomorph” cells have a sheet-like
periplast (e.g. Figs 7, 8); furthermore, the rhizostyle is non-keeled in the former and
keeled in the latter (Hoef-Emden & Melkonian 2003 and references therein). The fl agellar
ornamentation is an additional ultrastructural difference between the genera Cryptomonas
and Campylomonas – on which the cryptomorph and campylomorph concepts are based,
respectively – although it has gone unmentioned (Hoef-Emden & Melkonian 2003); it
follows the normal “2+1” pattern of mastigonemes in Cryptomonas, whereas there is
a single row of mastigonemes on each fl agellum in Campylomonas (Gromov et al. 1998;
see also Novarino 2003). The Campylomonas fl agellar ornamentation is also found in
Chilomonas (see Novarino 2003 and references therein), a similar genus apart from the
fact that it is secondarily heterotrophic rather than photosynthetic.
Dimorphism is known with absolute certainty in three species out of some 429
described ones. Out of a total of at least 74 culture strains examined in that respect,
only six – i.e. just over 8% – have been shown to be dimorphic (Hill & Wetherbee
1986; Hoef-Emden & Melkonian 2003). The respective signifi cance of the two morphs
is either unknown (Hoef-Emden & Melkonian 2003) or they are thought to represent
a haploid and a diploid stage, respectively, but no evidence of sexual reproduction has
been found, nor has the actual mechanism of transition from either morph to the other
been observed (Hill & Wetherbee 1986; Hoef-Emden & Melkonian 2003). Based on this
partial evidence, an hypothesis has been made whereby the co-existence of crypto- and
Table 3. Cryptomonads species known to exist both in “cryptomorph” and “campylomorph” form.
Species Reference
Dimorphic
strains
found
Crypto-campylomorph
transformation
actually observed
Signifi cance attributed
to crypto-campylomorph
transformation
Cryptomonas curvata Hoef-Emden
& Melkonian 2003
no no ?
C. pyrenoidifera Hoef-Emden
& Melkonian 2003
yes no ?
Proteomonas sulcata Hill & Wetherbee
1986
yes no Diploid and haploid sta-
ges; sexual reproduction
not observed
G. Novarino: Cryptomonad taxonomy in the 21st century 15
campylomorph cells within single cryptomonad species is very widespread, to the ex-
tent that it is the rule rather than the exception within the cryptomonads (Hoef-Emden
& Melkonian 2003).
The general applicability of the crypto-campylomorph hypothesis to the crypto-
monads as a whole deserves further investigation owing to its potential interest and
the fact that it is hitherto undemonstrated. However, by analogy to similar situations
in other groups of microalgae/protists it does not represent a signifi cant obstacle to the
continuing use of periplast characters for taxonomic purposes in the cryptomonads.
Heteromorphic life-cycles involving a modifi cation of morphological structures which
are accorded a taxonomic value are also known in other groups of microalgae/protists,
but this has not caused a major re-think of the taxonomic value of the structures in
question. For instance, species of coccolithophorids may have heteromorphic life-cycles
in which the various cellular stages bear morphologically different coccoliths (Young
et al. 2003) and were assigned originally to separate taxa. An example is Helicosphaera
carteri, whose life-cycle is now known to include a heterococcolith stage (on which
the original taxonomic description was based), plus a holococcolith stage which was
originally described as a species in its own right in a separate genus, i.e. Syracolithus
catilliferus (Young et al. 2003). Analogous situations also exist in other haptophyte taxa,
e.g. the occurrence of cell types with different organic scales within single species of
Chrysochromulina (Billard 1994). While of all this has prompted coccolithophorid and
haptophyte taxonomists to be aware of the possibility that different morphologies may
co-exist within single species, it has not caused the presence and morphology of coc-
coliths and organic scales to be abandoned as taxonomic characters. On the contrary,
morphologically-based taxonomic atlases and investigations continue to fl ourish (e.g.
Cros & Fortuño 2002; Young et al. 2003; Probert et al. 2007).
In summary, while the crypto-campylomorph hypothesis offers much scope for further
research the use of periplast features for taxonomic purposes is best maintained owing
to its proven usefulness for characterizing genera and species. However, the possibility
should also be borne in mind that a newly observed species with crypto- or campylomorph
features could actually represent an alternative morph of a different species, and perhaps
one which has been described already. In any case, it is likely that dimorphism may
be lost during culturing (Hoef-Emden & Melkonian 2003), which begs the question of
whether or not that might not also be the case in nature. Have present-day non-dimorphic
species lost the dimorphic condition sometime in the evolutionary past? Or have the
dimorphic species acquired the dimorphic condition later in their evolutionary history?
In other words, is dimorphism (where it exists) an ancestral condition, or a derived one?
These kinds of questions deserve to be investigated in depth before the generalised ap-
plicability of the crypto-campylomorph hypothesis can be accepted unquestionably, as
has happened already (e.g. Metfi es & Medlin 2007).
From a purely taxonomic point of view the crypto-campylomorph hypothesis has
led to a proposal to consider Campylomonas and Chilomonas as taxonomic (= subjec-
tive = heterotypic) synonyms of the genus Cryptomonas (Hoef-Emden & Melkonian
2003). This proposal has not found unanimous agreement and these genera continue to
be referred to by their original names in the general literature (e.g. Boechat et al. 2005;
16 Phycological Reports: Current advances in algal taxonomy
Scherwass et al. 2005; Cadotte et al. 2006; Sacca et al. 2008; Vaerewijck et al. 2010) as
well as on the Web (e.g. eBiodiversity 2008–2010; BioLib 2010; Micro*Scope 2010).
This contrasting opinion is based on the fact that Chilomonas is a secondary heterotroph
(osmotroph) with very distinct ecological requirements compared to the photosynthetic
genus Cryptomonas; on these grounds it continues to be maintained as a separate genus,
especially in ecologically-based taxonomic accounts (Novarino 2011).
The challenge of ecology
Cryptomonads are extremely widespread in marine and freshwater ecosystems world-
wide, particularly in the plankton, and have also been found in terrestrial subsurface or
subaerial habitats e.g. soil, groundwater and snow (see references in Novarino 2003).
Unsurprisingly therefore, the study of cryptomonad ecology has received a good deal
of attention; much of the available information is scattered in the specialist literature
but fortunately it was summarised in two comprehensive reviews (Klaveness 1988,
1988/1989). Basic information on the relationships between cryptomonad populations,
seasonality and the main physico-chemical environmental parameters, is relatively well
understood (Klaveness 1988, 1988/1989).
What is much less understood is how the various ecological factors affect individual
species, both from the point of view of the species’ preferences for and adaptations to
a given set of factors in relation to other species (niche partitioning), and that of the
ability of the environment to infl uence a species’ morphology, or in other words the
possibility that the phenotype might be partly environmentally-dependent. Such ques-
tions are pivotal in a discussion ultimately aimed at the concept of what constitutes
a cryptomonad “species”.
The mechanisms thanks to which niche partitioning is achieved must be quite re-
fi ned if they are to explain how, in one and the same environment, morphologically
similar species are able to co-exist simultaneously (as found for instance by Chang
1983 in the sea and Caljon 1987 in freshwaters), succeed one another regularly in time
(as found by Cronberg 1982 in freshwaters), or co-exist in time but without necessarily
being all present at all times (as found by Hada 1974 and Cerino & Zingone 2006 in
the sea; and Willén et al. 1980 and Khondker 2007 in freshwaters). There is relatively
little information on the subject, but species-specifi c differences in vertical migratory
behaviour are a documented mechanism for species to achieve spatial niche partitioning
and consequently co-exist. A fi eld study investigating the vertical migratory behaviour
of two morphologically similar species showed that they adjusted their depth based on
their optimum light intensity, which differed between them by a factor of 2.5 (Sommer
1982). Experimental studies have also confi rmed that cryptomonad species may behave
very differently when migrating vertically, and do so also in relation to optimum tempera-
ture and nutrients (Arvola et al. 1991) in addition to light (Clegg et al. 2003). Another
mechanism of spatial niche partitioning is given by the preference for low dissolved
oxygen concentrations. This was shown for Cryptomonas undulata, a freshwater species
whose type-habitat is the oxic/anoxic boundary layer (chemocline) near the lake bottom
(Gervais 1997a). In this environment low-light adapted photosynthesis (Gervais 1998)
G. Novarino: Cryptomonad taxonomy in the 21st century 17
may occur, and an ability to switch partly to heterotrophic (but not phagotrophic) nutri-
tion (Gervais 1997b) would also be advantageous. Interestingly, Cryptomonas undulata
was also found to co-exist simultaneously with Cryptomonas phaseolus and often also
Cryptomonas rostratiformis, thanks to short-term changes in the light climate near the
chemocline (Gervais 1998).
Although it is well known that ecological factors may infl uence the morphology
of a range of microalgae/ protists (see references in Kim et al. 2003), there is scant
information on the environment’s ability to affect the cryptomonad phenotype. A wide
morphological variability spectrum has been documented in nature in a small number
of species; however, cause-effect relationships between environmental factors and cell
morphology have not been addressed (Lund 1962; Javornicky 1976; Willen et al. 1980).
Culture observations on the presence of electron-dense bodies under the periplast of
Chroomonas grown at very high irradiance (Novarino & Lucas 1993a), and the non-motile
cells seen in ageing cultures of many cryptomonad species (Pringsheim 1944), suggest
that such cause-effect relationships do exist. More recently, a mesoscosm study (Kim
et al. 2003) showed that the posterior tail of a cryptomonad identifi ed as Plagioselmis
prolonga var. nordica shortened in mesocosms stocked with planktivorous silver carp
(Hypophthalmichthys molitrix Val.) and elongated by more than 50% in mesocosms
from which silver carp were removed. Such a fi nding is interesting also because it begs
indirectly the question of the function of the posterior tail of Plagioselmis, which is
a diagnostic feature for the genus. So far the tail function has been the object of only
one hypothesis, i.e. its intervention in cell coupling prior to sexual reproduction (Kug-
rens & Lee 1988).
In summary, even the scant evidence available so far suggests that cryptomonads
have evolved sophisticated and complex niche partitioning mechanisms. Furthermore,
their natural morphological variability could be related at least in part to direct or indirect
environmental effects. These topics deserve urgent attention, particularly in relation to
defi ning the concept of the cryptomonad “species”.
The challenge of molecular genetics
Beginning in the early 1990’s an ever increasing amount of molecular genetic information
on cryptomonads has become available. Initially the focus was mostly on the phylogeny
of the cryptomonads as a whole (Maerz et al. 1992; Douglas & Murphy 1994; McFadden
et al. 1994; Gilson et al. 1995; Liaud et al. 1997; Wang et al. 1997; Van der Auwera
et al. 1998; Douglas & Penny 1999), but more recently molecular sequence data have
also been used to investigate the phylogeny of cryptomonad genera and supra-generic
groupings. However, the situation is still far from resolved (Cavalier-Smith et al. 1996;
Marin et al. 1998; Deane et al. 2002; Hoef-Emden et al. 2002; Shalchian-Tabrizi et al.
2008). A detailed discussion of cryptomonad molecular phylogeny is outside the scope
of this paper; for a summary view the reader is referred to the most recent nuclear SSU
rDNA tree (Hoef-Emden 2008a) which, in addition to the Goniomonas clade (a primarily
heterotrophic genus lacking a nucleomorph), includes 6 clades defi ned also by phycobilin
pigments and the position of the nucleomorph. Inevitably, newly acquired molecular
18 Phycological Reports: Current advances in algal taxonomy
phylogenetic data will have an impact on existing classifi cation systems (Tables 4–6), but
so far no formal classifi cations based strictly on molecular criteria have been proposed.
[N.B. Cryptomonads are “ambiregnal” organisms falling under the dual jurisdiction of
the Botanical and Zoological Codes of Nomenclature, and as such they have been the
object of zoological classifi cations as well as botanical ones, as shown in Tables 4–6;
see also Novarino & Lucas 1993b, 1995].
Molecular sequencing data have also been used to investigate relationships between
species within some genera, and in some cases they have also been incorporated in spe-
cies- or genus-level taxonomic diagnoses (Deane et al. 1998; Hoef-Emden & Melkonian
2003; von der Heyden et al. 2004; Hoef-Emden 2007, 2008b; Lane & Archibald 2008;
Martin-Cereceda et al. 2010; see also Cerino & Zingone 2007 for a general review of
cryptomonad molecular studies). Comparative molecular investigations of two or more
taxa aimed at assessing whether or not their taxonomic separation is justifi ed are to be
encouraged, particularly if the use of molecular sequence data introduces a greater amount
of objectivity in taxonomy by quantifying the amount of genetic divergence necessary for
a taxon to be described as a separate species, genus and so forth. For instance, in other
microalgae/protists percentage similarity values of SSU rRNA genes between fi ve species
of the haptophyte genus Phaeocystis have been shown to range from 0.962 to 0.996,
representing an absolute number of nucleotide differences between 7 and 67 (Zingone
et al. 1999). Information of this kind constitutes a useful guideline for future taxonomic
investigations within Phaeocystis and likely also in the haptophytes as a whole.
Unfortunately however, scant information of this kind is available as yet in the
cryptomonads. A comparative study of nuclear and nucleomorph 18S rRNA sequences
in the genera Hanusia and Guillardia (Deane et al. 1998) revealed 14 nuclear and 264
nucleomorph nucleotide differences. In a comparative study of nuclear SSU rDNA in
Cryptomonas marssonii and C. pyrenoidifera from Korea (Kim et al. 2007), the value
of interspecifi c pairwise divergence was 0.6%. In other cases molecular information has
been used more in a qualitative way. In a number of Cryptomonas species, qualitative
molecular information (such as various unique combinations of sequence “motifs” in the
nucleomorph SSU rRNA) has been incorporated in formal taxonomic diagnoses, while
detailed information on sequences in certain helices of nuclear and nucleomorph SSU
rRNA have been included in a proposal for a revised generic diagnosis (Hoef-Emden
& Melkonian 2003). Together with the nucleomorph genome size, which ranged from
560 to 600 Kb, qualitative molecular information on the location of 18S and 5S rDNA on
the nucleomorph chromosomes has been incorporated in formal diagnoses of four newly
described Hemiselmis species otherwise characterized using elementary morphological
information revealed by LM only (Lane & Archibald 2008).
In summary, molecular data present cryptomonad taxonomy with a challenge which
deserves to be investigated in further detail in order to understand the overall signifi cance
of its impact, especially because molecular data have not yet provided a 100% objective
taxonomic method, be it in the cryptomonads or other microalgae/protists. This agrees
well with views whereby the application of molecular analyses to taxonomy should
not be assumed a priori to be free of subjectivity. Different taxonomists assessing the
results of a given molecular analysis might well reach different taxonomic conclusions
G. Novarino: Cryptomonad taxonomy in the 21st century 19
Table 4. Light microscopy based classifi cation systems of cryptomonads (Class Cryptophyceae) under the Botani-
cal Nomenclature. F = family, SC = sub-class.
System Subclass Orders Families Main characters
Pascher 1913 Eucryptomona-
dinae
Cryptomonadales Cryptomonadaceae Motile vs. non-motile
cells (SC) ; fl agella
position (F)
Nephroselmidaceae
Phaeocapsinae
Pascher 1914 Cryptomonadales Thallus organisation
Phaeocapsales
Cryptococcales
Cryptotrichales
Oltmanns 1922 Cryptomonadales Cryptomonadaceae As Pascher
Nephroselmidaceae
Cryptochrysidaceae
Phaeocapsaceae
Pringsheim 1944 Cryptomonadaceae Nutrition type,
fl agella position
Cryptochrysidaceae
Nephroselmidaceae
Katablepharidaceae Sk.
Cyatomonadaceae fam. nov.
Huber-Pestalozzi
1950
Monomastiginae Follows Pringsheim
with some
modifi cations
Cryptomonadinae Cryptomonadales Cryptomonadaceae
Cryptochrysidaceae
Senniaceae Sk. Instead of
Nephroselmidaceae
Katablepharidaceae Sk.
Cyatomonadaceae Prings.
Christensen 1962 Cryptomonadales Cryptomonadaceae Basically follows
Pringsheim
Katablepharidaceae Sk.
Planonephraceae fam. Nov.
instead of Nephroselmi-
daceae
Cyathomonadacea
Butcher 1967 Cryptomonadaceae Features of the
furrow-gullet system
Hemiselmidaceae fam. nov.
Hilleaceae fam. nov.
Bourrelly 1970 Cryptomonadales Cryptomonadacées Follows Pascher’s
basic concept
Hilleacées
Planonephracées
Katablepharidacées
Cyatomonadacées
Pleuromastigacées
20 Phycological Reports: Current advances in algal taxonomy
(Silva 2008). This is particularly true if molecular data are not used quantitatively, for
instance by comparing percentage similarity values of rDNA or other genes in two or
more taxa. It is also worth bearing in mind that – at least in other microalgae/protists –
variation in rDNA sequences could also be the result of neutral mutations (implying no
phenotypic changes or, even more importantly, functional ones: Fenchel 2005), which
begs the question of their generalised usefulness in taxonomic as opposed to phylogenetic
investigations. However, at least in some protists there are also indications that the high
genetic divergence of the slowly evolving rRNA gene may be refl ected by phenotypic
variation (e.g. Scheckenbach et al. 2006).
The cryptomonad “species”
The defi nition of the species concept in biology had preoccupied scientists for more
that 200 years at the time when Mayr (1957) edited a seminal work on the subject.
More than 50 years on, the study of the “species problem” continues, although it is not
necessarily producing generally applicable solutions (e.g. Wheeler & Meier 2000). With
so much still to discover about the biology, ecology and phylogeny of cryptomonads,
addressing the vast topic of what constitutes a cryptomonad species may appear pre-
mature. However, the cryptomonad species concept is central to any discussion on the
taxonomy of the group.
A major source of confusion encountered while addressing the species problem is
given by the scant attention which has been paid to “the two totally different meanings
of the word species when referring either to species as taxa or to the category species
(Mayr 2000b)”. Therefore, it is necessary to distinguish between the two uses of this
word (Mayr 2000b). Emphasis will be placed here on the fi rst meaning, by identifying
various criteria enabling taxonomists to delimit species-level cryptomonad taxa. This
approach, which is fundamentally distinct from (and much less complex than) defi ning
the concept of the category species (Mayr 2000b), is referred to here as the operational
aspect of the species concept. Table 7 lists a selection of mostly operational species
concepts, with defi ning criteria derived from a bibliographic analysis and the author’s
System Subclass Orders Families Main characters
Butschliellacées
Tetragonidiales Tetragonidiacées
Starmach 1974 Identical to Bourrelly
Christensen 1980 Cryptomonadales Cryptomonadaceae Basically follows
Bourrelly
Hilleaceae
Planonephraceae
Katablepharidaceae
Cyatomonadaceae
Pleuromastigaceae
Tetragonidiales Tetragonidiaceae
Table 4. Continued
G. Novarino: Cryptomonad taxonomy in the 21st century 21
own practical experience. Whatever the particular concept being followed, the common
element to all is the existence of a discontinuity of some sort between a given species
and any other one to which the same concept applies.
The biospecies. Perhaps the single most used species concept in biology is Mayr’s
biological one. However, this can only be applied to organisms which reproduce sexu-
ally (Mayr 2000a, 2000b) and it is very unclear if this is the case in the cryptomonads.
There are only two reports of sexual reproduction, consisting in fortuitous observations
of cell fusion in fi eld specimens of Chroomonas acuta and Cryptomonas sp. (Wawrik
1969, 1971), which have never been repeated again. The only other suggestions that
it may exist derive from an inference based on the hypothesized alternation of diploid
and haploid phases in cultures of Proteomonas sulcata (Hill & Wetherbee 1986), and
ultrastructural observations on karyogamy (Kugrens & Lee 1988) in cultured material of
a cryptomonad identifi ed as Chroomonas acuta (likely not the same cryptomonad studied
by Wawrik 1971), none of which have been repeated further. The case of Chroomonas
acuta sensu Kugrens and Lee (1988) is interesting owing to the fact that both cell mat-
ing types were produced in a single clonal culture (Kugrens & Lee 1988). In any case,
the fact that none of these observations have ever been repeated suggests that sexual
reproduction in the cryptomonads, where present, is a very rare event, and as such it is
of little consequence towards trying to apply the biospecies concept to the group. Even
when the existence of very rare sexual processes is allowed for, by and large cryptomonads
Table 5. Light microscopy based classifi cation systems of cryptomonads under the Zoological Nomencla-
ture. F = family, SO = sub-order.
System Class Orders or Suborders Families Main characters
Calkins 1909 „Phytofl agellata” Chrysofl agellida Pigmentation („yellow
chromatophores”)
Dofl ein 1909 „Phytofl agellata” Chromomonadina,
SO Cryptomonadina
?
Claus et al. 1932 „Phytofl agellata” Chromomonadina Cryptomonadidae ?
Grassé 1952 Cryptomonadines ?
Hall 1953 ? Cryptochrysidae Furrow-gullet system,
fl agellar insertion
Cryptomonadidae
Nephroselmidae
Lepsi 1965 ? Cryptomonadidae As Hall 1953
Nephroselmidae
Kudo 1966 ? Phaeocapsina (SO) Motile vs. non-
motile (SO), fl agellar
insertion (F)
Eucryptomonadina SO) Cryptomonadidae
Nephroselmidae
de Puytorac
et al. 1987
? Cryptomonadidae ?, fl agellar insertion,
nutrition
Planonephridae
Cyathomonadidae
22 Phycological Reports: Current advances in algal taxonomy
Table 6. Electron microscopy based classifi cation systems of cryptomonads under the Botanical and/or Zoological Nomenclature. F = family, O = order.
System Code Class (higher classifi cation in parenthesis) Orders Families Main characters
Cavalier-Smith
1989, 1993
ICZN (Kingdom Chromista, phylum Cryptista)
Cryptomonadea Cavalier-Smith – –
–
Goniomonadea Cavalier-Smith – –
Novarino and
Lucas 1993
ICBN Cryptophyceae orthogr. et diagnosis emend.
Novarino & Lucas
Goniomonadales
stat. et nom. nov.
Pyrenomonadales
ord. nov.
Cryptomonadales
ord. nov.
Goniomonadaceae Hill
Pyrenomonadaceae fam. nov.
Cryptomonadaceae sensu Butcher
Hemiselmidaceae Butcher
Presence/absence and position
of nucleomorph (O), fl agellar inser-
tion (F)
Corliss 1994 ICZN (Kingdom Chromista, phylum Cryptomonada)
Cryptomonadea Cavalier-Smith – –
Follows Cavalier-Smith
Goniomonadea Cavalier-Smith – –
Clay et al.
1999
ICBN (Phylum Cryptophyta)
Cryptomonadea Cavalier-Smith Cryptomonadales
Pyrenomonadales
Cryptomonadaceae
Campylomonadaceae
Pyrenomonadaceae fam. ?
Hemiselmidaceae fam. ?
Geminigeraceae fam. nov.
Chroomonadaceae fam. nov.
Presence/absence and position of
nucleomorph (O), fl agellar insertion
(F), phycobilins
Goniomonadea Cavalier-Smith Goniomonadales Goniomonadaceae Hill
G. Novarino: Cryptomonad taxonomy in the 21st century 23
will form clones rather than populations, and clones maintain their genotype from one
generation to the other by not interbreeding; therefore, they do not require isolating
mechanisms to protect their genotype (Mayr 2000a).
The phylospecies. Among the other available operational concepts, the phylospe-
cies sensu Wheeler and Platnick – defi ned based on “a unique combination of character
states in comparable individuals” (Wheeler & Platnick 2000) – appears promising in the
era of molecular sequencing studies because nucleotides offer a remarkable number of
character state combinations thanks to which it may be possible to diagnose species in
the organisms under study. This concept could be thought to apply, for instance, to the
Cryptomonas species which have been sequenced for nuclear and nucleomorph SSU
rRNA (Hoef-Emden & Melkonian 2003).
The morphospecies. Within the cryptomonads the morphospecies, which is largely
understood in a purely typological sense (Novarino & Lucas 1993b), is still the most
widely applied species concept. Despite the fact that in the last couple of decades an
increasing number of morphospecies have been described also on the basis of EM in-
formation, the overwhelming majority of cryptomonads have been described using LM
only. Inevitably, such a morphospecies concept is subject to a degree of subjectivity
because the delimitation of species boundaries will depend to an extent on the opinion
of the taxonomists making or refuting the formal descriptions, especially when the
descriptions are based on subtle differences visible (or, in many cases, hardly visible)
using LM. The “statistispecies” – a less subjective extension of this concept, in which
the existence of morphological discontinuities between species is tested statistically, be
it between the species’ respective nomenclatural types or living individuals in nature or
in cultures – has not been adopted widely. Anton and Duthie (1981) analysed sixteen
Cryptomonas species using cluster analysis, showing that binary characters were more
effective than continuous ones for discriminating between species. In a few other studies,
Table 7. A selection of species concepts (mostly understood in a purely operational sense) and their defi ning
criteria.
Concept Abbreviated name Defi ning criterion
Nominal species nomispecies Existence of a Linnean binomen, distinct from bino-
mina of other nomispecies
Morphological: typological morphospecies Existence of a morphologically-characterized
nomenclatural type, distinct from the nomenclatural
types of other morphospecies
Morphological: biometric/
statistical
statistispecies Existence of a statistically signifi cant discontinuity
between the variability range of given morphological
features in one statistispecies compared to others
Biological sensu Mayr biospecies Existence of a group of actually or potentially inter-
breeding populations, reproductively isolated from
other such groups
Phylogenetic sensu Wheeler phylospecies Existence of smallest possible aggregations of popu-
lations or lineages diagnosable by a unique combina-
tion of character states in comparable individuals
Ecological (including
physiological and biogeographic)
ecospecies Existence of a unique ecological niche, distinct from
the niche of other ecospecies
24 Phycological Reports: Current advances in algal taxonomy
basic statistical parameters were determined in a number of morphological characters
accorded taxonomic signifi cance at the species level (e.g. Novarino 1991a, 1991b; No-
varino & Lucas 1993a).
The ecospecies. The defi ning criterion of the ecospecies concept is the existence
of a unique ecological niche, which is distinct from the niche of other ecospecies. As
such it may appear to be somewhat different from the ecospecies concept of Klaveness
(1985), whereby an ecospecies is a distinct entity deriving from a pre-existing continuum
by means of ecological factors acting differently on different parts of the continuum
itself. However, the two interpretations are actually similar because the existence of
ecological gradients and combinations of particular ecological factors will always lead
to the existence of particular niches, which are potentially available for suitably adapted
species to occupy (Odum & Barrett 2004). As defi ned here, the ecospecies concept also
includes the physiological and biogeographical species concepts, whereby discontinui-
ties between species are achieved, respectively, thanks to differences in physiological/
biochemical/growth requirements and biogeographic distribution. The latter aspect ap-
pears particularly interesting in the light of the present debate on cosmopolitan versus
restricted geographic distribution in the microorganisms (e.g. Foissner 1999; Finlay 2002;
Fenchel & Finlay 2004). Based on available records, as a rule cryptomonads appear to
be widely distributed geographically but in at least a few cases restricted geographic
distributions may also apply, e.g. at least a few species have been noticed only in tropi-
cal habitats (Menezes & Novarino 2003).
Overall, the ecospecies concept appears to offer a somewhat more objective defi ning
criterion compared to other concepts because it is based ultimately on the ecological
principle of competitive exclusion, whereby two or more distinct species must have
distinct ecological niches in order to co-exist in space and time (Huston 1994). In order
to be able to apply this concept to the cryptomonads, it must be shown that species
recognizable using some other criterion (e.g. morphological or genetic) also occupy well-
defi ned, distinct niches, as studied for instance in the various species of Cryptomonas
already mentioned (Gervais 1997a, 1997b, 1998).
The nomispecies. Ultimately however, any elaboration on what constitutes a cryp-
tomonad “species” must rely on a preliminary estimate of how many species, however
defi ned, have been described formally in the literature (whether or not in accordance
with the Rules of Nomenclature), and this is the essence of the nomispecies concept. It is
often stated anecdotally that about 200 cryptomonad nomispecies exist (e.g. Hill 1991c),
or just over 100 limited to the Cryptomonadales (Lane & Archibald 2008). A closer
examination of source material (Novarino, unpublished) suggests that the true number
is at least 429 (Table 8). Even when the likely percentage of synonyms is allowed for
(estimated by the author at ca. 30%), the resulting fi gure for “good” nomispecies (i.e.,
those which are not nomenclatural or taxonomic synonyms of others) is between 2 and
3 times higher than the anecdotally quoted ones.
In spite of the wide availability of on-line databases with catalogues of species names
(often with accompanying descriptions and other information, e.g. Guiry and Guiry 2010),
there is as yet no offi cial requirement under either the Botanical or the Zoological Code
of Nomenclature that species names be registered in a centralised, publicly and freely
G. Novarino: Cryptomonad taxonomy in the 21st century 25
available repository. This would make it all that much easier to estimate nomispecies
numbers, not only in the cryptomonads but in all living organisms. Until such a require-
ment will be adopted, ultimately investigators wishing to gain insight on how many and
which species have been described in any particular group will have to rely on individual
initiatives. An example within the cryptomonads is Novarino & Gaddini (2010), which
includes a catalogue of nomispecies in the course of being populated and updated.
Unfortunately however, the rate at which the information becomes publicly available is
variable because the absence of fi nancial support for the website considerably limits the
possibility of relying on professional staff to mount the necessary data on-line.
In summary, no single operational species concept applies particularly better than
the others to the cryptomonads at present. However, by and large the morphospecies
concept has been the most widely used one. In the future, cryptomonad species de-
fi ned using a particular operational concept might have to be re-defi ned using a single
concept which is universally applicable to all cryptomonads and takes into account all
possible operational concepts. This will require an integrated study of single species
using morphological, ultrastructural, molecular, ecological, physiological and geographic
information at the same time. From an operational point of view, such an approach will
be useful in order to improve estimates of the number of existing cryptomonad species
(i.e., described + undescribed ones) based on scientifi c considerations. Existing estimates
have relied mostly on informed opinion – “guess-estimates” on the part of taxonomic
Table 8. Numbers of nomispecies in the cryptomonad genera.
Genus Nomispecies
Campylomonas Hill 1
Chilomonas Ehrenb. 30
Chroomonas Hansgirg sensu auctorum 80
Chroomonas Hansgirg sensu stricto 2–?
Cryptochloris Schiller 1
Cryptomonas Ehrenb. 200
Falcomonas Hill 1
Geminigera Hill 1
Guillardia Hill & Wetherbee 1
Hanusia Deane et al. 1
Goniomonas von Stein 3
Hemiselmis Parke 20
Hillea Butcher 10
Komma Hill 1
Plagioselmis Butcher emend Novarino et al.10
Proteomonas Hill & Wetherbee 3
Pyrenomonas Santore 10
Rhinomonas Hill & Wetherbee 10
Rhodomonas Karsten 40
Storeatula Hill 1
Teleaulax Hill 3
Total ≥ 429
26 Phycological Reports: Current advances in algal taxonomy
specialists (Andersen 1992: 1200 species) – rather than factual considerations. An al-
ternative albeit outdated estimate has been based on the ratio between the numbers of
undescribed morphospecies and total (= described + undescribed) ones found during
a newly conducted fi eld survey (Novarino 2005). Based on a value of 0.45 for this ratio
and the revised fi gure of 429 nomispecies mentioned above, a revised estimate of some
780 existing (morpho)species is suggested here, or about 550 when a synonymy rate
of 30% is allowed for.
Conclusions: Towards the next two hundred years
“What’s to come is still unsure.”
(William Shakespeare 1602, published 1623 – Twelfth Night,
Act II, Scene III: O Mistress mine, where are you roaming? )
Once the envisaged integrated approach to studying cryptomonads will have been
achieved, eventually it might become possible to investigate the concept of the category
species in the cryptomonads – i.e., the species as the unit of evolution. For that purpose,
suggested topics for future research might also include the following:
What are the relationships between diversity (e.g. morphological and/or molecular) –
and geographic distribution?
What are the relationships between diversity (e.g. morphological and/or molecular) –
and ecology, particularly in relation to niche partitioning?
Is the dimorphic condition an ancestral or a derived one? –
How, how frequently and in how many genera does sexual reproduction take –
place?
What is the function of various structures peculiar to cryptomonads, e.g. the furrow- –
gullet system, the periplast, and the posterior “tail”? Does each of these structures
have an adaptive value? Or have these structures evolved and been maintained
through neutral evolutionary mechanisms?
Until then, however, with anywhere between 100 and 350 cryptomonads still to be
discovered there is much need for descriptive alpha-taxonomy even if not all the desir-
able observational (e.g., EM) and analytical (e.g., molecular) techniques are available
during investigation, because biological classifi cation relies entirely on the ‘descriptive
basement level’ of biology, which is ‘the foundation on which all else is built’ (Mayr
1974). Without descriptive taxonomy to provide the basic building blocks, our knowl-
edge of cryptomonads and their evolution, biology and ecology will always be sketchy
at best.
Finally, during fi eld surveys reliable identifi cations documented in as much detail as
possible are desirable. Unfortunately, no comprehensive guide to all known cryptomon-
ads has been produced to date, although an attempt has been made to convey as much
relevant information as possible to non-specialist investigators, with a view to facilitat-
ing taxonomic identifi cation using what equipment is available to them at any one time
(Novarino 2003). The continued expansion and development of this approach on the
G. Novarino: Cryptomonad taxonomy in the 21st century 27
Web (Novarino & Gaddini 2010) will depend largely on the availability of sponsorship
and general support from interested parties.
ACKNOWLEDGEMENTS. I thank the organizers of the 29th Conference of the Polish Phycological Society
in Kraków-Niedzica in May 2010 for their kind invitation to deliver the talk on which this paper is
based. Further thanks to Prof. Konrad Wołowski also for his patience in receiving this manuscript.
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