A new genus, Helgoeca gen. nov., for a nudiform choanoflagellate

ARTICLE IN PRESS European Journal of

PROTISTOLOGY European Journal of Protistology 44 (2008) 227–237 www.elsevier.de/ejop

A new genus, Helgoeca gen. nov., for a nudiform choanoflagellate Barry S.C. Leadbeatera,, Ruhana Hassana,1, Michaela Nelsonb, Martin Carrb, Sandra L. Baldaufb,2 a

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Department of Biology, University of York, Heslington, York YO10 5YW, UK

b

Received 29 September 2007; received in revised form 17 January 2008; accepted 19 January 2008

Abstract A new genus, Helgoeca gen. nov., has been designated to accommodate a nudiform loricate choanoflagellate (American Type Culture Collection strain ATCC 50073) that was incorrectly attributed to the tectiform genus Acanthoecopsis ( ¼ Acanthocorbis). The first indication that this species might be nudiform came from a four-gene phylogeny of the choanoflagellates which recovered ATCC 50073 within a strongly supported monophyletic clade comprising two other nudiform taxa. Fortunately an isolate of the species in question was available from the ATCC and when observed in rapidly growing culture it was immediately apparent that this species divided with the production of ‘naked’ motile cells; a typically nudiform character. The beaker-shaped lorica of this species consists of an outer layer of approximately 11 longitudinal costae, which terminate anteriorly as spines, and an equal or larger number of helical costae, with a left-handed conformation, each of which terminates anteriorly adjacent to the base of a spine. The pattern of costae in this species is indistinguishable from that of Acanthocorbis nana Thomsen and for this reason A. nana has been transferred to the new genus Helgoeca gen. nov., as the type species. r 2008 Elsevier GmbH. All rights reserved. Keywords: Choanoflagellate; Nudiform replication; Lorica-construction; Molecular phylogeny; Helgoeca gen. nov.; Acanthocorbis unguiculata

Introduction Loricate choanoflagellates are a universal protistan component of marine environments. Their distinctive feature is the basket-like lorica that consists of a pattern of siliceous costae composed of rod-shaped costal strips Corresponding author. Tel.: +44 121 427 1930; fax: +44 121 414 5925. E-mail address: [email protected] (B.S.C. Leadbeater). 1 Current address: Department of Aquatic Sciences, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS), Malaysia. 2 Current address: Department of Evolutionary Biology, Uppsala University, Norbyva¨gen 18D, S752 36, Uppsala, Sweden.

0932-4739/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2008.01.003

attached to each other end-to-end. Individual loricae usually comprise a system of outer longitudinal and inner transverse or helical costae, although there are many variations on this theme (Thomsen and Buck 1991). Loricae are assembled in a single continuous movement lasting a few minutes from accumulated groups of costal strips on the surface of the cell (Leadbeater 1979a, b, 1994). Assembly involves a combined forward and left-handed rotational movement of strips that is mediated by the cell cytoskeleton and a special group of actin-based tentacles (Leadbeater 2008; Leadbeater et al. 2008). In contrast to the constancy of the choanoflagellate cell, which is spherical to ovoid with a single anterior

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flagellum surrounded by a collar of tentacles, the lorica displays considerable variation. The ecological success of loricate species to colonise a wide range of microniches within the marine environment can, to a large extent, be related to the subtle variations in lorica form, size and costal pattern (Thomsen and Buck 1991). At present two distinctive groups of loricate choanoflagellates can be distinguished on the basis of the timing of costal strip production in the cell-cycle and the morphology of cell division (Manton et al. 1981). One group displays nudiform replication, whereby a cell with a lorica divides to produce a ‘naked’ motile cell which swims away from the parent lorica, settles on to a surface, produces a set of costal strips and then assembles its lorica (Leadbeater 1979a, 2008). The other group displays tectiform replication, whereby a parent cell with a lorica produces costal strips in advance of division, stores them at the top of the collar and when a complete set has been produced the cell divides, inverts and the juvenile is pushed out of the parent lorica with the accumulated strips. Once free of the parent, the juvenile assembles its lorica (Leadbeater 1979b, 1994). Currently, only five nudiform species attributable to three genera, Acanthoeca, Polyoeca and Savillea, have been recognised, in comparison with over 100 tectiform species. In a recent four-gene phylogenetic study of choanoflagellates involving two nudiform and four tectiform species, one of the latter, designated ‘Acanthoecopsis unguiculata’ deposited at the American Type Culture Collection as strain ATCC 50073, regularly appeared in the nudiform clade. To confirm the tectiform status of this isolate, a culture was acquired from the ATCC and subjected to microscopical study. Quickly it was realised that this species was, in fact, nudiform. Furthermore, electron microscopy revealed that it was similar to another already described tectiform species, Acanthocorbis nana Thomsen in Thomsen et al. (1997). Clearly these findings raise major nomenclatural problems which need to be addressed before further confusion ensues. To accommodate another species within the nudiform clade it has, for reasons presented below, been necessary to create a new genus, Helgoeca gen. nov. The nomenclatural problem outlined here has also cast some doubt on the authenticity of certain other species attributed to Acanthocorbis Hara and Takahashi and whilst no other adjustments are made at present, the significance of the nudiform and tectiform modes of division to the systematics, evolution and ecology of choanoflagellates in general is considered.

Nomenclature The genus Acanthoecopsis Norris has had one of the most complicated and troubled of nomenclatural

histories experienced within the Choanoflagellida. The generic name was originally designated by Norris (1965) for a long-stalked loricate choanoflagellate with a crown of spines that resembled in most respects the closely related genus Acanthoeca Ellis, 1929. Norris called the type species Acanthoecopsis spiculifera Norris. Contemporaneously, Boucaud-Camou (1966) observed a similar species which she attributed to Polyoeca dichotoma Kent, 1880. Leadbeater (1972) designated a second Acanthoecopsis species, A. apoda Leadbeater, on account of the similarity of the crown of spines and other features of the lorica chamber to those of the type species. Thomsen (1973) added a third species A. unguiculata Thomsen. This was followed by further additions by Hara and Takahashi (1984) and Thomsen et al. (1997). Meanwhile the similarity of the type species of Acanthoecopsis to Polyoeca dichotoma Kent was reviewed by Leadbeater (1979a) and finally recognised nomenclaturally by Hara and Takahashi (1984) who declared Acanthoecopsis spiculifera Norris synonymous with Polyoeca dichotoma Kent and designated a new genus Acanthocorbis Hara and Takahashi, with the type Acanthocorbis (Acanthoecopsis) apoda (Leadbeater) Hara and Takahashi, for the remaining species of Acanthoecopsis. As currently construed Acanthocorbis now contains 10 species, A. apoda (Leadbeater, 1972) Hara and Takahashi, 1984, A. unguiculata (Thomsen, 1973) Hara and Takahashi, 1984, A. nana Thomsen, 1997, A. weddellensis Thomsen, 1997, A. prolongata Thomsen, 1997, A. haurakiana Thomsen, 1981, A. camarensis Hara, 1996, A. tintinnabulum Marchant, 1981, A. campanula (Espeland, 1986) Thomsen, 1991 and A. asymmetrica (Thomsen, 1977) Hara and Takahashi, 1984.

Materials and methods The choanoflagellate culture known as ‘Acanthoecopsis unguiculata’ was obtained from the American Type Culture Collection as strain ATCC 50073. This species was isolated from a seawater sample collected from Lower Narragansett Bay, RI, USA and was deposited with the ATCC by Prof. Paul G. Davis in 1981. In Birmingham the ‘bacteria-containing’ culture was maintained at 5 1C in sterile seawater with autoclaved rice grains to provide the organic enrichment necessary for bacterial growth. For SEM, circular coverslips were placed in small Petri dishes filled with seawater without enrichment. A small inoculum of cells was added and the sample left for 2 weeks. Coverslips were then removed and without drying were placed in a Petri dish with several drops of osmium tetroxide on the lid. After 2 min exposure to

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osmium tetroxide vapour, the coverslips were removed, passed through a standard acetone series (5 min for each concentration) and following treatment with absolute acetone, the coverslips were placed in a critical-pointdryer and flash dried using liquid CO2. Dried samples were placed on stubs, sputter-coated with gold and viewed using a Philips XL-30 FEG ESEM. Light microscopy was carried out using a Leitz Ortholux microscope fitted with interference contrast objectives. Images were recorded using a digital camera. The type micrograph of Acanthocorbis nana originally published in Thomsen et al. (1997) is reproduced here as Fig. 10 and a light micrograph as Fig. 2. The TEM image of Acanthocorbis unguiculata (Fig. 11) was supplied by Professor Helge Thomsen and the SEM image of Acanthocorbis unguiculata published by the Australian Antarctic Division (Fig. 12) was supplied by Dr. Harvey Marchant. Genomic DNA was obtained from six loricate species – ATCC 50073, Acanthoeca spectabilis Ellis, Diaphanoeca grandis Ellis (ATCC 50111), Diplotheca costata Valkanov, Savillea micropora (Norris) Leadbeater and two strains of Stephanoeca diplocostata Ellis, one from French coastal waters (ATCC 50456) and one from Australian coastal waters – and three non-loricate species – Monosiga brevicollis Ruinen (ATCC 50154), Table 1.

229

M. ovata Kent (supplied by Prof. Peter Holland) and Salpingoeca pyxidium Kent (ATCC 50929). Fortymillilitre cultures were centrifuged at 4000 rpm for 40 min and the resultant pellets treated following the NaCl/ethanol protocol of Carr et al. (2006). Two ribosomal DNA genes (SSU and LSU) and two protein-coding genes, a-tubulin (tubA) and 90-kDa heat shock protein (hsp90), were employed for phylogenetic reconstruction. New sequences generated for this study are listed later in Table 3. The following SSU sequences were obtained from GenBank: AF084234 (Diaphanoeca grandis, 1794 bp), AF084230 (Monosiga ovata, 1765 bp) and L10823 (Acanthoecopsis unguiculata (ATCC 50073), 1780 bp). Initial SSU PCR fragments were amplified using the primers of Medlin et al. (1988). Individual sequencing reactions were then performed on this using the primers listed in Table 1. LSU sequences were amplified using the primer pairings NLF108/18+NLR1126/22, NLF1105/22+NLR2098/24, NLF1999/18+NLR2871/ 19 and NLF2551/21+NLR3535/22 taken from the European Ribosomal RNA Database. Hsp90 and tubA sequences were amplified using the primers listed in Table 1. PCR products for LSU, hsp90 and tubA were ligated into the pGEM-T Easy Vector (Promega) and transformed into Subcloning Efficiency DH5a

PCR primers designed for choanoflagellate species included in Fig. 14

Gene

Primer

Annealing temperature (1C)

Nucleotide sequence

SSU

300F 528F 640F 1055F 1200F 300R 536R 690R 1055R 1200R

53 50 45 54 50 58 57 45 54 52

50 -AGGGTTCGATTCCGGAG-30 50 -CGGTAATTCCAGCTCC-30 50 -YAGAGGTGAAATTCT-30 50 -GGTGGTGCATGGCCG-30 50 -CAGGTCTGTGATGCTC-30 50 -TCAGGCTCCCTCTCCGG-30 50 -GWATTACCGCGGCKGCTG-30 50 -AGAATTTCACCTCTG-30 50 -CGGCCATGCACCACC-30 50 -GGGCATCACAGACCTG-30

tubA

atF1 atF3 atR1 atR2 atR3

50 58 58 58 50

50 -GGGCCCCAGGTCGGCAAYGCNTGYTGG-30 50 -CTCAGGGGNAARGCAGAYGC-30 50 -CACCAGGTTNGTYTGRAAYTC-30 50 -GCGCATAACCTCNCCNACRTACCA-30 50 -AAGGCCTTCNCCYTCYTCCAT-30

hsp90

HSP1F HSP2F HSP3F HSP4F HSP1R HSP2R HSP3R HSP4R HSP-loricate-F HSP-Dc-F HSP-Dc-R

55.7 55.7 50 50 55.7 55.7 50 50 50 50 50

50 -CCAGCCTACGAC(TCN/AGY)AAYAARGAR-30 50 -GACATCAGCATGATCGGNCARTTYGGN-30 50 -ACGACCGCGTCAARCTNTAYGTN-30 50 -AAGCTGGGGATHCAYGARGAY-30 50 -CCATCTTCCRTCNACYTCYTCCAT-30 50 -CCGCGCYTTCATBATYCKYTCCAT-30 50 -AACAGCGTACTCRTCBATNGGNTC-30 50 -CTCGCAGTTRTCCATDATRAANAC-30 50 -CTCAAGGACTACGTCACCCGC-30 50 -TGGGAGGATCATCTTGCCGT-3 50 -CTGCATCCTTAGTTTCACCAG-3

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Chemically Competent Cells (Invitrogen). Plasmid DNA was extracted using Qiagen’s Spin Miniprep kit and sequenced using T7 and SP6 primers (Macrogen Inc., Seoul, Korea). Combining our sequences with publicly available sequences from GenBank produced complete datasets for both rRNA genes as well as hsp90. Sequences for tubA could not be amplified from ATCC 50073 and the French population of Stephanoeca diplocostata. The two absent genes, as well as absent nucleotides from individual genes, were classed as missing data in our phylogenetic analyses. It is well established that phylogenetic reconstruction is quite insensitive to missing data (Philippe et al. 2004; Philippe and Telford 2006). Furthermore, our single gene phylogenies are in broad agreement (i.e. there are no strongly supported incongruencies) both with each other and with the concatenated phylogeny (data not shown), therefore the absence of these two genes is not expected to have any impact on our phylogenies. Each gene was individually aligned in ClustalX (Thompson et al. 1997) and then edited by eye. The four-gene alignments were concatenated to make a single 6404 bp dataset, which was partitioned so that the ribosomal genes were modelled separately from the protein coding genes. Within the protein coding genes, first and second positions were modelled separately from third positions. The alignment was run through Modeltest 3.7 (Posada and Crandall 1998), which determined that the GTR+I+G model (Rodrı´ guez et al. 1990) was the most appropriate for phylogenetic analysis. A Maximum Likelihood (ML) tree was produced in Phyml 2.4.4 (Guindon and Gascuel 2003), employing a GTR+I+G model with a four-category gamma distribution. The ML tree was bootstrapped with 1000 replicates. A Bayesian phylogeny was produced using MrBayes 3.1.1 (Ronquist and Huelsenbeck 2003). The MCMC analysis ran with one cold and three hot chains for 250,000 generations with a sampling frequency of 10. The first 25% of sampled trees were discarded as burn-in. All alignments are available from the authors upon request.

Taxonomic descriptions Helgoeca Leadbeater gen. nov. Diagnosis Cells solitary with a beaker-shaped lorica comprising two layers of costae. Inner layer contains 10–12 helical costae, with left-handed direction of coiling, arranged regularly around the lorica chamber; each costa terminates anteriorly adjacent to the base of the longitudinal strip forming a spine at the anterior end of a longitudinal costa. Outer costal layer consists of

10–12 regularly spaced longitudinal costae which extend from the base of the lorica and terminate anteriorly as a ring of spines. Division is nudiform with production of a flagellated juvenile cell for dispersal. Sedentary spindleshaped juvenile produces costal strips and assembles lorica. The name Helgoeca has been chosen to acknowledge Professor Helge Thomsen as the original authority of Acanthocorbis nana and also to celebrate his considerable contribution to the study of choanoflagellates. Type species: Helgoeca (Acanthocorbis) nana (Thomsen) Leadbeater.

Helgoeca (Acanthocorbis) nana (Thomsen) Leadbeater comb. nov. Diagnosis Cells solitary, usually sedentary often as part of a biofilm or clump of bacteria and detritus. Lorica beakerto funnel-shaped 7–10 mm long  4–5 mm wide, comprising two layers of costae. Inner layer contains 10–12 helical costae, with left-handed direction of coiling, arranged regularly around the lorica chamber; each costa terminates anteriorly adjacent to the base of the longitudinal strip forming a spine at the anterior end of a longitudinal costa. Outer costal layer consists of 10–12 regularly spaced longitudinal costae which extend from the base of the lorica and terminate anteriorly as a ring of spines. Division nudiform. Marine and brackish water; universal in distribution. Type micrograph: Fig. 6 in Thomsen et al. (1997), reproduced here as Fig. 10. Type locality: A brown ice sample from Weddell Sea (Thomsen et al. 1997). Subjective synonym: ATCC 50073. Observations When observed with light microscopy in the living condition, individual cells are located within a closefitting lorica which is relatively thick and has regular lateral dot-like markings (Figs 1 and 2). The anterior crown of spines widens slightly towards the anterior end, the dot-like markings continuing for about half the length of the spines. Cell division is preceded by withdrawal of the parent flagellum. Following division the juvenile cell, with a long flagellum, is located immediately above the daughter cell that remains within the parent lorica (Fig. 3). The juvenile cell swims away (Fig. 4), settles on to a surface, becomes spindle-shaped and produces a covering of costal strips (Fig. 5). Scanning EM of whole cells reveals the arrangement of costae. Unfortunately, in most specimens there is considerable damage with separation and relocation of strips, particularly around the lorica chamber (Figs 6–8). However, by studying many specimens it is possible to reconstruct a reasonably consistent image. There are

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1

2

3

6

4

7

5

8

Figs 1–8. Light microscopy and SEM of ATCC 50073. Fig. 1. Light micrograph of interphase cell showing beaker-shaped lorica. Bar ¼ 2 mm. Fig. 2. Copy of light micrograph of Acanthocorbis nana from Thomsen et al. (1997). Bar ¼ 2 mm. Fig. 3. Light micrograph of the products of a recent division. The upper juvenile cell has a long flagellum. Bar ¼ 2 mm. Fig. 4. Spindle-shaped juvenile cell with flagellum. Bar ¼ 1 mm. Fig. 5. SEM of spindle-shaped juvenile cell surrounded by bundles of costal strips. Bar ¼ 1 mm. Figs. 6–8. SEM of loricae showing general arrangement of outer longitudinal and inner helical costae. Bar ¼ 1 mm. Each arrow in Figs 6 and 8 denotes the point at which the anterior end of a helical costa joins a corresponding longitudinal costa. In Fig. 7 the smaller arrow denotes one helical costa terminating on a single longitudinal costa; the larger arrows denote two helical costae terminating on a single longitudinal costa.

two layers of costae, an outer arrangement of 10–12 longitudinal costae that run from the bottom of the lorica and terminate anteriorly as spines. The costal strip forming the projecting spine is subtended by one or two strips with substantial overlaps (Figs 6–8 and 9a). The location and alignment of the strips comprising the longitudinal costae on the surface of the lorica chamber are often severely disrupted, although occasionally the vertical nature of the costa can be seen (Figs 6 and 7). The inner lorica layer comprises a system of regularly spaced helical costae, usually the same in number as the longitudinal costae, that terminate in a regular manner adjacent to the bases of the corresponding longitudinal strips forming the spines (Figs 6 and 8 (arrows), 7 (smaller arrow) and 9a). In some instances two costal strips, presumably from two helical costae, appear to terminate at the base of a single spine (Fig. 7, larger arrows). In most specimens the pitch of the helical costae flattens anteriorly (Figs 6–8). The direction of coiling of the inner helical costae is always left-

handed; this refers to the fact that they ascend in a clockwise manner when viewed from the flagellar pole (Fig. 8).

Comparison of ATCC 50073 with two species of Acanthocorbis and Saepicula pulchra Table 2 lists the dimensions and other salient details relating to the lorica of four choanoflagellates of similar appearance. Apart from a few minor details ATCC 50073 and Acanthocorbis nana are identical. The type micrograph of Acanthocorbis nana published in Thomsen et al. (1997) is reproduced here as Fig. 10 and comparison with the loricae illustrated in Figs 6 and 7 confirms the similarity. Thomsen et al. (1997) recorded Acanthocorbis nana as being tectiform but this was probably because of the similarity of this species to others of the genus (e.g. Acanthocorbis apoda) that are known to be tectiform (Thomsen, personal communication).

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Whilst Acanthocorbis nana superficially resembles Acanthocorbis unguiculata (compare Fig. 10 with Figs. 11 and 12), there are some important matters of detail that differ. In particular each spine of A. unguiculata terminates in a pointed claw (Fig. 11 inset) and in general the strips of this species are slightly thicker. Whilst there is a 1:1 ratio between the helical and longitudinal costae in both species (compare Figs 10 and 12), nevertheless the location of the anterior junction between the two costal systems differs in the two species. Whereas in Acanthocorbis nana the anterior end of a helical costa terminates at the overlap between the anterior spine and the subtending longitudinal costal strips (Figs 9a and 10, arrows), the equivalent junction in Acanthocorbis unguiculata is some distance below this overlap (Figs 9b and 12, arrows). Additionally, the general appearance of the helical costae and the shape of the lorica chamber differ between the two species (compare Figs 6–9 with Figs 11 and 12).

Saepicula pulchra has the obvious difference of having an anterior ring (Fig. 13) but, apart from this feature, it is similar in general costal arrangement and lorica shape to Acanthocorbis unguiculata even including each spine terminating in a pointed claw (Fig. 13 inset).

Molecular phylogeny Two four-gene phylogenies, constructed using Bayesian inference and Maximum Likelihood protocols, were based on two ribosomal DNA genes and two protein-coding genes. Details of the GenBank accession numbers and sequence lengths for the various genes sampled during this study are recorded in Table 3. The phylogenies were made up from loricate and nonloricate choanoflagellates and the loricate representatives were recovered as a monophyletic clade in both phylogenies (Fig. 14). Within this clade, the nudiform and tectiform taxa were each recovered as strongly supported monophyletic groups (1.00 Bayesian inference Posterior Probability and 100% Maximum Likelihood Bootstrap Support). ATCC 50073 appears as a sister group to Savillea micropora and Acanthoeca spectabilis within the nudiform lineage. Within the tectiform clade, Stephanoeca diplocostata, which has helical costae as well as rings to hold the longitudinal costae in place, forms a sister group to Diplotheca and Diaphanoeca both of which have only rings holding their longitudinal costae in place.

Discussion

Fig. 9. Diagram illustrating the respective locations at which an individual helical costa attaches to the corresponding longitudinal costa in ATCC 50073 (a) and Acanthocorbis unguiculata (b). Note that in ATCC 50073 the helical and longitudinal costae join at the overlap between the anterior spine strip and the subtending strips. In A. unguiculata the helical and longitudinal costae join below the region of overlap.

The most important conclusion to emerge from the information presented here is that ‘Acanthocorbis (Acanthoecopsis) unguiculata’ strain ATCC 50073 is a nudiform loricate choanoflagellate and not a tectiform species as its name would imply. A secondary outcome has been that ‘Acanthocorbis (Acanthoecopsis) unguiculata’ strain ATCC 50073 is closely similar in terms of lorica shape and construction to Acanthocorbis nana Thomsen. Both of these outcomes, in their different ways, highlight the difficulty of identifying small cells with close-fitting, beaker-shaped loricae with anterior

Table 2. Cell dimensions and lorica characters of ATCC 50073, Acanthocorbis unguiculata (Thomsen) Hara and Takahashi, A. nana Thomsen and Saepicula pulchra Leadbeater Cell character

ATCC 50073

Acanthocorbis nana Thomsen

Acanthocorbis unguiculata Thomsen (Hara and Takahashi)

Saepicula pulchra Leadbeater

Lorica dimensions (mm) Spines/longitudinal costae (number) Division

7–10  4–5 10–11 Nudiform

10  5 10–14 ?

16  5 11 Tectiform

10–13  5–6 10 Tectiform

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10

12

233

11

13

Figs 10–13. Fig. 10. Copy of the type micrograph of Acanthocorbis nana Thomsen. Reproduced with permission from Thomsen et al. (1997). Arrows denote abuttal of the anterior ends of the helical costae on base of spines. Bar ¼ 1 mm. Fig. 11. Acanthocorbis unguiculata (Thomsen) Hori and Takahashi. Empty lorica showing general arrangement of costae. Bar ¼ 1 mm. Fig. 11 inset. Top of a spine showing characteristic clawed tip. Bar ¼ 50 nm. Fig. 12. A. unguiculata. SEM of lorica showing the general arrangement of costae. Arrows denote abuttal of the anterior ends of the helical costae on base of spines. Reproduced with permission from Marchant et al. (1987). Bar ¼ 1 mm. Fig. 13. Saepicula pulchra. TEM of lorica showing arrangement of costae including anterior ring. Bar ¼ 1 mm. Fig. 13 inset. Anterior tip of longitudinal costa showing clawed tip similar to that of Acanthocorbis unguiculata. Bar ¼ 0.1 mm.

spines. Even where information is available from electron microscopy, inadvertent mistakes can easily be made (Marchant et al. 1987). With the benefit of hindsight, clarity in identification could only have been achieved if the mode of cell division had been known and the costal construction of the lorica fully understood. The absence of groups of costal strips at the top of the collar could also have been an indicator of the nudiform condition although this feature is unsatisfactory because of its negative nature and many tectiform cells lack accumulations of costal strips if they are in the stationary phase of growth. For good measure the availability of SSU and LSU sequence data would have

been desirable. None of this information was available at the time strain ATCC 50073 was identified and deposited at the ATCC in 1981. A nomenclatural alteration is now essential to remedy the situation. ‘Acanthocorbis (Acanthoecopsis) unguiculata’ strain ATCC 50073 and Acanthocorbis nana Thomsen must be combined into one taxon and transferred to the nudiform clade. The question that arises is whether Acanthocorbis nana can be incorporated into an existing nudiform genus or whether it is necessary to designate an entirely new genus especially for the purpose. This matter can only be resolved by a consideration of the lorica morphology of the three

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Table 3.

B.S.C. Leadbeater et al. / European Journal of Protistology 44 (2008) 227–237

Sequences generated for this study

Species

SSU

Acanthoeca spectabilis Diaphanoeca grandis Diplotheca costata ATCC 50073 Monosiga ovata Salpingoeca pyxidium Savillea micropora Stephanoeca diplocostata (Australia) Stephanoeca diplocostata (France)

EU011922 – EU011923 – – EU011930 EU011928 EU011926 EU011927

LSU (1864 bp) (1885 bp)

(1824 bp) (1772 bp) (1820 bp) (1818 bp)

EU011933 EU011939 EU011938 EU011934 EU011940 EU011946 EU011944 EU011938 EU011943

tubA (3752 bp) (3534 bp) (3225 bp) (2680 bp) (3315 bp) (3571 bp) (2010 bp) (3225 bp) (3502 bp)

EU011962 EU011966 EU011965 – EU011967 EU011971 EU011970 EU011969 –

hsp90 (936 bp) (787 bp) (936 bp) (936 bp) (936 bp) (756 bp) (754 bp)

EU011949 EU011954 EU011953 EU011950 EU011956 EU011960 EU011959 EU011961 EU011955

(765 bp) (1404 bp) (1809 bp) (504 bp) (1527 bp) (1434 bp) (781 bp) (516 bp) (516 bp)

GenBank accession numbers are shown in bold and sequence lengths are stated in base pairs (bp). Two ribosomal DNA genes (SSU and LSU) and two protein-coding genes, a-tubulin (tubA) and 90-kDa heat shock protein (hsp90), have been sequenced.

Fig. 14. Molecular phylogeny of choanoflagellates based on a concatenated four-gene data set. The tree was derived by Bayesian inference based on a combination of tubA, hsp90 and SSU and LSU rDNA nucleotide sequences. Branches are drawn to scale and the bar represents the number of nucleotide substitutions per site. Bayesian inference posterior probabilities and maximum likelihood bootstrap percentage values are given above and below branches, respectively.

current nudiform genera (Savillea, Acanthoeca and Polyoeca). Table 4 lists the more important lorica characters of the three nudiform genera and ATCC 50073. All have two layered loricae based on longitudinal and helical costae. In Savillea the lorica is probably the simplest in

construction and consists of an inner layer of regularly spaced helical costae with an outer layer of longitudinal costae (Leadbeater 2008). The basic relationship between the two types of costae (helical to longitudinal) is 1:1 although in S. micropora the ratio can be as high as 1:4. In Polyoeca the lorica comprises a system of approximately 14 longitudinal costae that extend from the base of the stalk to the tip of the spines (Leadbeater 1979a). In the mid-lorica region, these costae may double or treble to provide additional support. On the inner surface of the lorica chamber there are one or more bands of horizontal helical costae. Within the crown of spines there are supporting strips with a lefthanded inclination. Acanthoeca is the most complex and derived of all the nudiform genera (Leadbeater 1979a, Leadbeater et al. 2008). Although similar and probably closely related to Polyoeca, the single layer of longitudinal costae forming the chamber comprises a closely-wound spiral. There is apparently no inner layer of costae which has allowed the outer costae in this instance to become helical, a pattern that in other loricate species is normally confined to the inner layer of costae. The anterior crown of spines is similar to that of Polyoeca. Some features of ATCC 50073 are also seen in Savillea and Polyoeca. The posterior part of the lorica of ATCC 50073 is not dissimilar to that of Savillea in that it consists of regularly spaced helical costae stabilised by an outer arrangement of longitudinal costae in a 1:1 or 2:1 relationship, helical to longitudinal costae (Leadbeater 2008). The anterior crown of spines of ATCC 50073, particularly the manner in which each helical costa terminates adjacent to the base of a spine, is reminiscent of an equivalent appearance in Polyoeca and Acanthoeca (Leadbeater et al. 2008). However, since in all cases the similarities are only partial, a new genus, Helgoeca gen. nov., has been designated for the new combination comprising ATCC 50073 and Acanthocorbis nana Thomsen. The latter becomes the type species

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Table 4.

235

Comparison of lorica characters typical of the three current nudiform genera and ATCC 50073

Lorica character

Savillea

Acanthoeca

Polyoeca

ATCC 50073

Lorica form Lorica chamber

Flask-shaped

Long-stalked or beaker-shaped One-layered – closely-wound helical costae

Long-stalked

Beaker-shaped

Two-layered – outer longitudinal costae and inner bands of helical costae Crown of spines with inner rings of strips

Two-layered – outer longitudinal and inner widely spaced helical costae

Anterior end of lorica

Two-layered – outer longitudinal costae and inner widely spaced helical costae Terminates in aperture surrounded by helical costae

Crown of spines with inner rings of strips

for the new genus and provides the specific epithet for the new combination. The number of extant nudiform species now stands at six (Leadbeater 2008; Leadbeater et al. 2008). Since there are lists of marine choanoflagellates from many locations around the world, it is unlikely that many other nudiform taxa will be found (see Thomsen et al. 1997; Leakey et al. 2002 for references). The four-gene phylogeny recovers all the nudiform species so far tested in a distinctive group indicating that they are an evolutionarily coherent assemblage and not a heterogeneous collection of taxa. All nudiform species share a similar mode of cell division with the production of a motile cell which swims away, settles down and produces costal strips (Leadbeater 2008; Leadbeater et al. 2008). In principle, this is similar to the sequence of events found in thecate choanoflagellates (Leadbeater 1977). The morphology of division in nudiform taxa is, at least superficially, less complex than that found in tectiform species where the juvenile cell is inverted and pushed into a covering of accumulated strips (Leadbeater 1979b, 1994). The production of costal strips by nudiform juveniles and the subsequent assembly of the lorica are, again, reminiscent of the course of events in thecate species where cell wall material is secreted into vesicles within the juvenile cell and then extruded to produce a theca for itself (Leadbeater 1977). The sequence of events in tectiform choanoflagellates once again appears more complex with the strips being produced prior to division and then passed on to the juvenile (Leadbeater 1979b, 1994). The most significant outcome that appears to be related to the difference between nudiform and tectiform division is that on the juveniles of nudiform taxa costal strips are arranged vertically, whereas on the juveniles of tectiform species they are arranged both vertically and horizontally. This latter organisation appears to be related to the production of transverse rings in tectiform species, a feature not observed in nudiform taxa (Leadbeater, unpublished observation).

Crown of spines with an inner helical costa terminating at base of each spine strip

If transverse rings are an exclusive feature of the tectiform lorica and are never found in nudiform species, how can there be confusions of identity between some nudiform and tectiform taxa? Species attributable to Acanthocorbis would appear to provide the answer to this question. Acanthocorbis unguiculata is superficially similar to ATCC 50073 which accounts for the original confusion over the naming of the latter. The similarity is because both species comprise a small cell with a closefitting lorica bearing an anterior crown of spines (compare Figs 7 and 12). The similarities also extend to lorica construction which contains an inner layer of regularly spaced helical costae and outer longitudinal costae in a 1:1 ratio (Marchant et al. 1987). Differences between the two taxa are subtle; in particular the anterior tips of the spines of A. unguiculata end in pointed ‘claws’ (Thomsen 1973). However, the ‘true’ A. unguiculata is unequivocally tectiform; cells have been observed with accumulations of costal strips at the top of the collar – a feature never observed in nudiform taxa. How, then, can the tectiform condition be unequivocally associated with transverse rings if a transverse ring does not feature on the lorica of A. unguiculata? The answer would appear to be that this species no longer produces transverse rings. To illustrate this point it is necessary to observe Saepicula pulchra (Fig. 13) which is similar in most respects to Acanthocorbis unguiculata (Figs 11 and 12) except that the anterior end of the lorica terminates in a transverse ring (Leadbeater 1980). In particular, the anterior tips of the longitudinal costae in S. pulchra terminate in pointed ‘claws’ just like those of Acanthocorbis unguiculata (compare Figs 11 inset and 12 inset). Thus Saepicula pulchra would appear to be A. unguiculata with an anterior transverse ring and vice-versa. The conclusion must be that Acanthocorbis, as currently construed, is a heterogeneous grouping of species all with anterior spines most of which are tectiform but some of which may be more closely related to other genera.

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A striking feature of the nudiform clade is how small it is in terms of species and how limited these species are in terms of ecological diversity. All six species are relatively small, sedentary and associated with biofilms or clumps of bacteria; there are no truly planktonic or free-floating representatives (Leadbeater 2008; Leadbeater et al. 2008). This is in striking contrast to tectiform species which number more than 100 and are amongst the most important components of the heterotrophic marine nanoplankton. It may be that the evolution of the transverse ring bestowed a mechanical advantage on the lorica, enabling the cell to produce a lighter more open costal framework thereby minimising the density of the cell whilst at the same time increasing its resistance to sinking. Whether or not the tectiform mode of costal strip accumulation and cell division was essential for this innovation is a matter of conjecture and will be discussed further in later communications.

Acknowledgements We are grateful to Professor Helge Thomsen for commenting on the manuscript in draft form and for supplying the three illustrations included as Figs 2, 10 and 11. We also thank Dr Harvey Marchant for supplying Fig. 12 and Mrs. Joyce Kent for her assistance with preparation of the script. Dr. Andrew Polaszek and Mr. Stephen Tracey of the Natural History Museum, London kindly gave advice on nomenclatural matters.

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