Loricate choanoflagellates (Acanthoecida) from warm water seas. IX. Coronoeca gen. nov., Polyfibula Manton and spiny forms of Parvicorbicula Deflandre

The ambition to generate an overview of warm water loricate choanoflagellate biodiversity, based on a classic morphometric approach, is here completed by analyses of a range of tiny forms with anterior spines or projections and in most cases also a posterior pedicel. The warm water study complements previously obtained results from the more extensively studied temperate and polar regions of the world’s oceans. It thus contributes to a significantly more balanced approach to global diversity patterns for these organisms. The current survey includes taxa such as Polyfibula elatensis, Parvicorbicula pedicellata, as well as a range of primarily undescribed and taxonomically challenging species, that are in an interim approach allocated to Coronoeca gen. nov. (C. kosmaniae sp. nov., C. conicella sp. nov., C. superpositus (Booth) comb. nov., C. marchantii sp. nov., C. tongiae sp. nov., and C. patongiensis sp. nov.). The analysis of warm water acanthoecid biodiversity has revealed in total 80 species from the six geographic regions sampled, corresponding to approximately 50% of all loricate species described. Nineteen species are previously undescribed forms. The Andaman Sea, Thailand, and West Australia are in a global context the most species-rich regions with 62 and 64 species respectively. 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).


Introduction
In an ongoing effort (Thomsen and Østergaard 2019a; Thomsen and Østergaard 2019b;Thomsen and Østergaard 2019c;Thomsen and Østergaard 2019d;Thomsen and Østergaard 2019e;Thomsen and Østergaard 2021;Thomsen et al. 2020a; Thomsen et al. 2020b) to provide a first comprehensive overview of warm water loricate choanoflagellate diversity, based on a traditional microscopical approach, we here primarily deal with a contingent of small, spiny and often pedicellated species. While a single species (i.e. Polyfibula elatensis (Thomsen, 1978) Manton in Manton and Bremer 1981), belongs to an unambiguously defined genus, the remaining taxa have unresolved phyloge-netic positions within the loricate choanoflagellate species matrix. It is in our opinion beneficial to future research, to be able to refer to a morphospecies by using a specific taxon name, rather than an ad hoc labelling of forms that will not to the same extent, systematically and rigorously, serve as a reference point for future observations of the specific taxon. We have therefore created the genus Coronoeca gen. nov., that can serve as an interim 'parking space' for these forms, while awaiting an in-depth analysis aided by state-of-the-art molecular tools. The generic type-species, C. kosmaniae sp. nov., has unique morphological features, that irrespective of the future fate of other species currently allocated to the genus (i.e. C. conicella sp. nov., C. superpositus (Booth, 1990) comb. nov., C. marchantii sp. nov., C. tongiae sp. nov., and C. patongiensis sp. nov.) will likely remain a phylogenetically valid unit. Parvicorbicula superpositus Booth, 1990, is transferred to Coronoeca (C. superpositus (Booth, 1990) comb. nov.) based on critical discrepancies, between this species and a core group species of Parvicorbicula as defined by Leadbeater (2015), relating to in particular mutual positions of transverse and longitudinal costae.
The current paper rounds off the survey of warm water loricate choanoflagellate diversity with a brief global perspective of the results obtained, and an inter-comparison of the geographic regions sampled.

Material and methods
The material that constitutes the background for this and a series of papers on warm water acanthoecid choanoflagellates was collected over a period of 35 years. The geographic origin of samples is recorded in Fig. 1. See Thomsen and Østergaard (2019a) for information on each of the collection sites and sampling campaigns. In order to substantiate the analysis of Coronoeca superpositus (=Parvicorbicula superpositus) we have added material (Fig. 12) from Danish coastal waters. In order to further corroborate observations from our standard regions (Thomsen and Østergaard 2019a) we have appended to this paper wherever relevant, also historical data from Japan (Seto Inland Sea) to species descriptions, as well as the global analysis of loricate choanoflagellate biogeography (Figs. 16,17).
The general protocol for processing water samples for light (LM) and (TEM/SEM) electron microscopy was according to Moestrup and Thomsen (1980) and Thomsen (1982). For details on sample processing, preparational issues and microscopes used see Thomsen and Østergaard (2019a). The Japanese material from the Shioya Coast (Seto Inland Sea, Kobe, Japan) comprises surface water samples collected from April 1979to September 1982 ; 27-33 PSU). The processing of the Japanese samples for electron microscopy followed the basic principles outlined by Moestrup and Thomsen (1980).
The material examined here is dried, which means that the natural 3-D structures have collapsed to become 2-D structures, leading to a partial dislocation of costal strips, and an artefactual expansion of in particular the lorica width. While several structures can still be measured with confidence, e.g. lorica height and the length of spines and pedicels, it does imply that certain values such as lorica diameter, typically at the level of the transverse costa(e), cannot be measured directly but only calculated from measurements of the circumference. This approach was used in the species descriptions below. A circular dot refers to a single spot sampling, while a line or square indicates that samples were collected along extended transects. For further information see the materials and methods section in Thomsen and Østergaard (2019a).
Efforts were made when describing lorica features to follow the terminology standards that have developed in the course of dealing with these organisms; see e.g. Leadbeater (2015;loc. cit. chapter 4 and glossary p. 278) and .
The unfortunate existence of an electron microscope specific problem causing negatives to appear horizontally flipped, was discussed in Thomsen and Østergaard (2019d). The evidence in favour of dealing with this prob-lem as a purely technical issue is overwhelming. Scans of the affected negatives have accordingly been flipped horizontally to produce non-inverted images. In the current publication this applies to: Fig. 2a; Fig. 3c; Fig. 6a;Fig. 8a,b;Fig. 14a,b;Fig. 15a,b,g. The shade matrix (Fig. 17) is constructed using the PRI-MER software package (ver. 7) that has a built-in wizard that carries out the sequences of routines needed to perform the analysis (Clarke et al. 2014;Clarke and Gorley 2015). Fig. 2. a-m. TEM (a, d-g) and LM whole mounts (b, c, h-m; phase contrast) of Polyfibula elatensis from the Gulf of California (a-c, e), the Andaman Sea, Thailand (d, f, g), the Caribbean Sea (h), the Sargasso Sea (i), West Australia (j, l, m), and the equatorial Pacific Ocean (k). (a, d) Complete cells; the arrow (a) points to a single facetted costal strip from the posterior transverse costa; (b, c, h-m) Selection of specimens from various geographic regions; (e-g) Detail (reversed printing) of distinctly (e) and less distinctly (f, g) facetted transverse costal strips; the arrows (f, g) point to median thickenings of costal strips. The scale bar (b) applies to all LM images.

Results and discussion
Polyfibula Manton in Manton and Bremer, 1981 Species of Polyfibula are characterized by small, barrelshaped loricae that comprise 6-8 longitudinal costae and three transverse costae. The transverse costae are all located inside the longitudinal costae. The individual species may have a posterior pedicel and short to medium-sized spines anteriorly. Most characteristically, the transverse costal strips that form the anterior and intermediate costal rings, are all furnished with centrally placed enlargements (facets) that anteriorly secures the fusion (T-joins) of longitudinal and transverse costal strips. A single costal strip from the posterior transverse costa may also be furnished with a median central facet ( Fig. 2a; arrow). The protoplast is located posteriorly in the lorica. The genus is characterized by tec-tiform division ( Fig. 2J; see also Manton and Bremer 1981;loc. cit. Figs. 2, 18, 31).
The genus currently comprises five formally described species (i.e. P. sphyrelata (Thomsen, 1973) Manton in Manton and Bremer, 1981;P. elatensis (Thomsen, 1978) Manton in Manton and Bremer, 1981; P. caudata (Leadbeater, 1975) Manton in Manton and Bremer, 1981;P. hexacostata Manton in Manton andBremer, 1981, andP. stipitata Manton in Manton andBremer, 1981). A comprehensive description of the genus is provided by Manton and Bremer (1981). Tong (1997a) advocated that P. caudata, P. hexacostata and P. stipitata should be relegated to synonyms of P. sphyrelata. The distinguishing lorica features are merely the number of longitudinal costae, and the presence or absence of a pedicel. Such lorica features are generally accepted to show some level of variability at the intra-specific level (P. elatensis (see below) typically has seven longitudinal costae, however occasionally eight; Fig. 3. a-j. TEM (a-e) and LM whole mounts (f-j; phase contrast) of Parvicorbicula pedicellata from the Andaman Sea, Thailand (a, b, d, e), the Gulf of California (c, i, j), the equatorial Pacific (f), and West Australia (g, h). (a-c) Complete loricae; (d) Detail (reversed printing) of anterior transverse costa (from (b)) showing the exterior position of the longitudinal costae; (e) Detail of the mid-lorica transverse costa (from (a)) to highlight the interior position of the longitudinal costae; (f-j) Selection of specimens from various geographic regions. The scale bar (g) applies to all LM images. P. sphyrelata usually has seven longitudinal costae (Thomsen 1973) but is also found with eight longitudinal costae ). It is thus likely that the species circumscriptions provided by Manton and Bremer (1981) are too narrow.
While three morphotypes (i.e. P. caudata, P. hexacostata, P. stipitata) are sparingly recorded and almost exclusively from North Pacific and North Atlantic subarctic seas (Manton and Bremer 1981), the generic type species P. sphyrelata is more widely reported from in particular mid-latitude locations on both hemispheres (Thomsen 1973;Moestrup 1979;Hallegraeff 1983;Tong 1997a;Tong et al. 1998;Bérard-Therriault et al. 1999;Menezes 2005). The species also extends into brackish waters (Thomsen 1979;Ikävalko and Thomsen 1997), Antarctic circumpolar waters (Marchant 1985;Thomsen et al. 1990), and is further recorded from the Beagle Channel, Patagonia (Thomsen, unpublished). P. elatensis (Thomsen, 1978) Manton in Manton and Bremer, 1981 (Fig. 2) Polyfibula elatensis is unambiguously distinguished from other species of Polyfibula based on the presence of prominent anterior spines (Fig. 2a, d), each one-half costal strip in length. The material examined here ( Fig. 2; Table 1) is morphometrically in good agreement with the type material from the Gulf of Aqaba (Thomsen 1978). While the facets on transverse costal strips stand out clearly in material from the Gulf of California (Fig. 2a, e), these structures are, however, in material from the Andaman Sea (Fig. 2d, f, g) often very little pronounced, and appearing as just a minor median bulging of the transverse costal strip (Fig. 2f,g;arrows). Such specimens are hard to distinguish from Parvicorbicula pedicellata ( Fig. 3; see further below). The vast majority of specimens examined have seven longitudinal costae. However, a few cells with eight longitudinal costae were also registered (e.g. Fig. 2h). While the type material (Thomsen 1978) was characterized by a posterior pedicel comprising just a single costal strip, it is evident that specimens examined here may occasionally, and most likely depending on environmental nuances, carry a considerably longer pedicel (Fig. 2j,l) that is up to five costal strips long.
Polyfibula elatensis was frequently observed at all sites here examined (Table 2) with the exception of the East Mediterranean Sea. It is also known from Japanese coastal waters and has furthermore been recorded from the Gulf of Aqaba, Israel (Thomsen 1978), Galapagos and N. Alaska (Manton and Bremer 1981), California , Sydney, Australia (Tong et al. 1998), Pettaquamscutt River Estuary, Rhode Island, USA (Menezes 2005), Brazil (Bergesch et al. 2008), and also from Beagle Channel, Chile, and Friday Harbor, Puget Sound, USA (Thomsen, unpublished), and from New Zealand (Moestrup, unpublished).

Parvicorbicula Deflandre, 1960
Non-spiny warm water forms of Parvicorbicula were commented on in Thomsen et al. (2020a), and overall concerns with reference to a proper circumscription of the genus discussed at length. The generic type species (P. socialis (Meunier, 1910) Deflandre, 1960 forms, together with a small group of species of Parvicorbicula and Pleurasiga Schiller, 1925, i.e. Pa. quadricostata Throndsen, 1970; Pa. circularis Thomsen, 1976; Pa. corynocostata Thomsen, Garrison and Kosman, 1997;Pl. minima Throndsen, 1970;Pl. reynoldsii Throndsen, 1970, Pl. tricaudata Booth, 1990, what appears to constitute a phylogenetically coherent cluster of species, that in the future should be singled out as a separate genus. A feature that characterizes this group of species is first and foremost the fact that they have both the anterior and the mid-lorica transverse costae on the outer surface of the lorica. Additional shared morphological features are longitudinal costae (4-10) composed of three costal strips, two transverse costae separated by a single longitudinal costal strip, anterior T-joins between longitudinal and transverse costal strips, a flattened and slightly biforked termination of the tip of the anterior longitudinal costal strips, and the presence of a membrane, suspended from the free posterior tips of the middle layer of costal strips, and enveloping the posteriorly positioned protoplast. None of the species have anterior spines or projections, and a pedicel is only reported from Pl. tricaudata.
The genus Parvicorbicula additionally comprises eight morphologically diverse species, including P. pedicellata (see below), that in different ways do not conform to the morphological circumscription of the core group of taxa referred to above. Because of uncertainties with respect to selecting, within the loricate choanoflagellate species matrix at large, more appropriate taxonomic positions for these outlier species, Thomsen et al. (2020a) opted for maintain- ing status quo while awaiting in particular molecular data to support the decisions needed. P. pedicellata Leadbeater, 1973 (Fig. 3) There is overall good agreement between the P. pedicellata material examined here ( Fig. 3; Table 1) and the type material from Bay of Kotor, Montenegro (Leadbeater 1973). The only noticeable difference is with respect to the number of longitudinal costae. Leadbeater (1973) reports that there are 6-9 longitudinal costae, whereas in our material there is consistently seven longitudinal costae. In P. pedicellata (Table 3) the anterior transverse costa is located inside the longitudinal costae ( Fig. 3d) while the mid-lorica transverse costa (Fig. 3e) is exterior. The posterior transverse costa is interior.
The only P. pedicellata morphological feature that tiesup this species to the Parvicorbicula core group of species as circumscribed above, is the fact the mid-lorica transverse costa is located outside the longitudinal costae. Features that separate P. pedicellata from Parvicorbicula sensu stricto are the presence of three transverse costae and anterior spines, and also the fact that each longitudinal costa consists of four costal strips.
Parvicorbicula pedicellata shares features with Coronoeca spp. (see below), e.g. anterior spines and four costal strips in each longitudinal costa, but is still clearly differentiated because of the external position of the mid-lorica transverse costa. While awaiting supporting molecular evidence it appears most relevant to make no taxonomic changes with reference to P. pedicellata.
Parvicorbicula pedicellata is easily mistaken for Polyfibula elatensis (Fig. 2). Decisive differences between these taxa, that can only be resolved using electron microscopy, are the presence in Pol. elatensis of facets in the middle of transverse costal strips, and the fact that all transverse costae are located inside the longitudinal costae in Pol. elatensis (Table 3). At the light microscopical level the best distinguishing feature is the overall lorica size (Pa. pedicellata is 25% smaller) and the shape of the lorica chamber, which is strictly conical in Pa. pedicellata ( Fig. 3f-j) while anteriorly more parallel sided in Pol. elatensis (Fig. 2b, c, h-m).
Diagnosis: Lorica conical or barrel-shaped and comprising 4-10 longitudinal costae and 2-4 transverse costae. There are anterior projections or spines, and the lorica may have a compound pedicel, a simple pedicel or no pedicel. Each longitudinal costa comprises four costal strips (including anterior projection). Anterior junctions between longitudinal and transverse costal strips can be either T-junctions or E-junctions. All costal strips are narrow rods. Minor morphological modifications can apply to anterior projections or costal strips from the pedicel. The longitudinal costae are external relative to the mid-lorica transverse costa. Tectiform division presumed but not yet verified in the type species. Generic type species: Coronoeca kosmaniae sp. nov. Etymology: Genus name derived from 'corona' (Latin) meaning 'crown' and referring to the prominent anterior spines or projections, and 'oicos' (Greek) meaning 'house'.
Coronoeca kosmaniae sp. nov. (Fig. 4a, 5, 6) Diagnosis: Lorica barrel-or bell-shaped (lorica height: 25.5 ± 1.2 mm incl. anterior projections and excl. posterior pedicel), constructed of rod-shaped costal strips, and with modified attenuating anterior projections (seven) that have a proximal swelling ca. 0.2 mm from the posterior tip (Fig. 5e, f). Anterior transverse costal strips attach to longitudinal costae at the level of the swellings creating T-junctions. The lorica chamber is composed of seven longitudinal costae and two transverse costae ( Fig. 5a-d). The anterior transverse costa rests in a fork created by the overlapping longitudinal costal elements (Fig. 5e, f) and is exterior relative to the tip of the penultimate longitudinal costal strip, but interiorly located relative to the anterior projection. The mid-lorica transverse costa is inside the longitudinal costae. A longitudinal costa is composed of four costal strips (including the anterior projections). Longitudinal costae converge posteriorly, with the posteriormost longitudinal costal strips being noticeably curved and sometimes duplicated. Transverse costae each comprising seven costal strips, and of approximately the same size (anterior ring: 15.5 ± 0.6 mm/mi d-lorica ring: 15.2 ± 0.7 mm). Lorica with a compound pedicel, usually with 1-3 diverging costal strips. Both anterior and posterior projections tapering, and ca. 1.0 mm longer than all other costal strips (7.2 ± 0.6 mm versus 6.2 ± 0.6 mm). The protoplast is located at the posterior end of the lorica chamber, within a suspensory membrane (Figs. 5c,d;6a,d). Tectiform division presumed but not verified from an accumulation of newly formed costal strips in the collar region. Holotype: The specimen illustrated in Fig. 5d  The spacious C. kosmaniae lorica has an uncomplicated and fairly standard lorica construction, but is nevertheless uniquely defined, in particular because of the costal strip swellings (Fig. 5e, f) at the base of the anterior projections, and at a point where the anterior transverse costa crosses. These localized swellings are reminiscent of the median facets on Polyfibula transverse costal strips (Manton and Bremer 1981;see also Fig. 2e) and also the less confined swellings in the posterior one third of anterior longitudinal costal strips in Saroeca attenuata (Thomsen 1979).
There is in C. kosmaniae a variable degree of costal strip duplication at the posterior lorica end. The TEM image (Fig. 6a) thus shows a lorica in which all posterior longitudinal costal strips are duplicated (or even triplicated; Fig. 6a arrows) except for one costal strip ( Fig. 6a; arrowhead). Duplications are also easily detected from LM (Fig. 6b). The anterior projections and costal strips from the compound pedicel are basically similar and deviate from midlorica longitudinal costal strips by being larger and attenuating in width from one end to the other (Fig. 5d). This precludes both the possibility that the posterior lorica end costal strip duplication could in part be explained by a displacement of costal strip elements from the compound pedicel, and likewise that the compound pedicel could be interpreted as a simple displacement of costal strips from the posterior lorica end duplications. A compound pedicel is a feature that is otherwise shared among several species, e.g. Didymoeca tricyclica (Bergesh et al. 2008), Pleurasiga tricaudata (Booth 1990;Thomsen et al. 2020a), Thomsenella cercophora (Thomsen and Boonruang 1983;Thomsen and Østergaard 2019e), and Syndetophyllum pulchellum (Thomsen and Moestrup 1983;Thomsen et al. 2020b).
The costal strip pattern at the level of the anterior transverse costa, where the transverse ring rests in a fork created by an overlap between neighbouring longitudinal costal strips (the ring is external relative to the penultimate longitudinal costal strip, however, overlaid externally by the anterior projection), is basically similar to what is observed in species of Diaphanoeca, e.g. Diaphanoeca grandis (Leadbeater 2015;loc. cit. page 80 and Figs 4.29-4.33) and Conion groenlandicum (Thomsen 1982), and also mirrored in species discussed below (C. conicella, C. superpositus, and C. marchantii).
Coronoeca kosmaniae is superficially reminiscent of Diaphanoeca throndsenii (Thomsen and Østergaard 2021). However, in this species there is consistently eight longitudinal costae, anterior E-junctions between longitudinal and transverse costal elements, no swelling at the posterior end of a projection, a pedicel that comprises a single costal strip, and no indication of costal strip duplications at the posterior lorica end.
The combination of lorica features in C. kosmaniae as accounted for above calls for the description of a new genus.
Coronoeca kosmaniae was frequently observed in samples from the Gulf of California, Mexico, and has also been observed in samples from the Seto Inland Sea, Japan. The Japanese specimens are morphometrically exactly similar to the Mexican type material.
Ignoring the anterior projections and the pedicel, C. conicella does superficially appear similar to e.g. Parvicorbicula circularis (Thomsen 1976). Shared features include four longitudinal costae each comprising three costal strips, and two transverse costae where the anterior costa has eight costal strips with every second strip forming T-junctions with the longitudinal costae, while the posterior transverse costa is formed by 5-6 costal strips. The decisive factor when considering a possible phylogenetic linkage between the two species is, however, that while the mid-lorica transverse costa is located outside the longitudinal costae in P. circularis it is inside in C. conicella (Table 3). This is a fundamental difference that is intimately linked to processes being part of the lorica assembly following cell division. Despite a superficial resemblance, P. circularis and C. conicella must therefore be kept apart at the supra-generic level.
In C. conicella the anterior transverse costa is external relative to the tip of the penultimate longitudinal costal strip, however, overlaid by the anterior projections (Figs. 7b, 8a, b). A similar set up is observed in C. kosmaniae (see above) and C. superpositus (see below).
Coronoeca conicella is, as defined here, a highly variable taxon, and has accordingly been lavishly illustrated from various geographic regions. We have at all sites examined observed two morphotypes of the species, i.e. forms with a short pedicel comprising just a single costal strip (e.g. Fig. 7c, k; 8j), and others that have an extended pedicel comprising multiple costal strips (e.g. Fig. 7d, g, j; 8f). There is an obvious linear linkage ( Fig. 10A; r 2 = 0.77) between lorica height and length of pedicel in the material examined (>60 specimens), as well as a non-patchy distribution, indicating that this element of variability is likely an inherent characteristic of the species. It can be further noticed from Fig. 10A that there are no obvious regional differences, although specimens from the Gulf of California are much less variable in size than e.g. specimens from West Australia.
Somewhat surprisingly it has been noticed that approximately 10% of all loricae examined have five rather than four longitudinal costae ( Fig. 7g; 8a, f). The Gulf of California (Mexico) has a marked overrepresentation of such forms with five longitudinal costae (Fig. 10B). In material from Japanese coastal sites the only morphotype observed has five longitudinal costae (Fig. 9a, 10B).
All costal strips are narrow rods and quasi-identical throughout the lorica. However, the anterior projections do differ in being anteriorly attenuating and pointed and markedly thickened posteriorly (Fig. 8b). The maximum diameter of the rod is at the crossing of the transverse costa. Costal strips from extended pedicels generally appear somewhat more compact than costal strips elsewhere.
Coronoeca conicella was observed at all sites sampled (Table 2) and the form with an extended pedicel also occurred in Japanese coastal samples. The species has previously been observed (specimens with five longitudinal costae) in samples from the Galapagos Islands, Peru (I. Manton ( †), pers. com.) and Sydney, Australia (Tong et al. 1998, loc. cit. Fig. 2l; referred to as Parvicorbicula superpositus).
Coronoeca superpositus (Booth, 1990) comb. nov. (Figs. 4d, e, 11, 12) Basionym: Parvicorbicula superpositus Booth, 1990 When first described (Booth 1990) this species was primarily discussed with reference to Parvicorbicula socialis (Meunier, 1910) Deflandre, 1960, with which it does share a striking resemblance (e.g. ten longitudinal costae, two transverse costae separated by a single longitudinal costal strip, anterior T-junctions), when ignoring the C. superpositus anterior projections and pedicel. However, a close inspection of the material from Danish coastal waters that have been included here to consolidate the C. superpositus species circumscription, clearly unveils that the mid lorica transverse costa is located inside the longitudinal costae (Table 3), while it is clearly exterior in Parvicorbicula socialis (see e.g. Buck 1981, loc. cit. Figs. 9, 11). This is perhaps even more evident when consulting the excellent SEM images of C. superpositus published by Bérard-Therriault et al. 1999;loc. cit. Pl. 142a, b). An exterior versus interior placement of the mid-lorica transverse costa is, as previously emphasized, a fundamental distinguishing characteristic, which is coupled to intrinsic processes of lorica assembly, and hence being a feature only applicable at the supra-generic level. The anterior transverse costa is exterior relative to the tips of the longitudinal costal strips from the tier below, however externally overlaid by the anterior projections (Fig. 11a, 12c). The costal strip configuration at the level of the anterior transverse costa is thus similar to what is observed in both C. kosmaniae and C. conicella. The actual morphological elaboration of the anterior projection is also identical in the species C. kosmaniae, C. conicella and C. superpositus. The projection is anteriorly sharply attenuated while bulging posteriorly (Fig. 12c). The maximum width is attained at exactly the level of the anterior transverse costa.
Specimens resembling C. superpositus (Fig. 11) are only sparingly observed in our warm water samples (Andaman Sea, Thailand). While the mid-lorica transverse costa is also in these specimens internal relative to the longitudinal costae, they deviate from C. superpositus sensu stricto by having fewer longitudinal costae (eight) and by the fact that all and not just a sub-set of longitudinal costae are here terminated by anterior projections (Fig. 11a, b). For the time being the variability encountered in Thailand specimens, when compared with C. superpositus sensu stricto (Fig. 12), is considered to represent intraspecific variability. The Thailand morphotype of Coronoeca superpositus was sparingly observed also in samples from Japanese coastal waters.
Coronoeca marchantii sp. nov. (Figs. 4h, 13) Diagnosis: Lorica funnel-shaped (lorica height: 14.2 ± 1.4 mm incl. anterior projections and excl. posterior pedicel) and con-structed of rod-shaped costal strips organized in (7)-8 longitudinal costae and three transverse costae separated by single tiers of longitudinal costal strips. There are anterior attenuating projections and a pedicel comprising a single costal strip. A longitudinal costa is composed of four costal strips (including the anterior projections). The anterior transverse costa (diam. 8.9 ± 0.8 mm) comprises as many costal strips as there are longitudinal costae (E-junctions). The mid-lorica transverse costa (diam. 6.6 ± 0.5 mm) has the same number of costal strips with much larger overlap between neighbouring strips. The posterior transverse costa (diam. 2.9 ± 0.4 mm) comprises 4-5 costal strips. All transverse costae are internal relative to the longitudinal costae. The anterior transverse costa rests in a fork created by overlapping longitudinal costal elements. It is exterior relative to the tip of the penultimate longitudinal costal strip, but interiorly located relative to the anterior projections (Fig. 13a). The protoplast is located at the posterior end of the lorica chamber, within a suspensory membrane (Fig. 13a). Tectiform division evidenced from the accumulation of costal strips in the collar region (Fig. 13e, h). Holotype: The specimen illustrated in Fig. 13a  There is little to add to the formal description of C. marchantii which has a well-defined costal strip configuration of its tiny conical lorica. In most cases this species can be unambiguously identified also from LM of dried specimens (e.g. Fig. 13c-e, j, k). A few slightly deviant forms were encountered in samples from West Australia and the Sargasso Sea (Fig. 13f-i). While these specimens obviously have eight longitudinal costae and three transverse costae, the difference noted is a slight pushing forward of the anterior transverse costa. Arrows (Fig. 13f-i) indicate where to find the transition between the anterior projections and the first tier of longitudinal costal strips. The overall appearance of these forms is reminiscent of Parvicorbicula pedicellata (Fig. 3). However, the pattern of crossing between longitudinal costae and costal strips from the anterior transverse costa (T-versus E-junctions) and the mere number of longitudinal costae (seven versus eight) adequately separates the two forms. For now, it thus appears relevant to treat this small deviation in lorica morphology as merely C. marchantii intraspecific variability. However, the validity of this approach will need to be tested using molecular tools.
Coronoeca marchantii was observed at all sites sampled with the exception of the eastern Mediterranean Sea (Table 2). It is additionally observed from the Galapagos Islands, Peru (I. Manton ( †), pers. comm.).
Coronoeca tongiae sp. nov. (Figs. 4f, 14a-g) Diagnosis: Lorica funnel-shaped (lorica height: 14.6 ± 0.9 mm) and constructed of rod-shaped costal strips organized in 9-10 longitudinal costae and four transverse costae. There are anterior spines up to one half longitudinal costal strip in length. A longitudinal costa is composed of four costal strips. The mid-lorica and posterior transverse costae are separated by single tiers of longitudinal costal strips. The three anterior transverse costae (diam. 12.0 ± 1.1 mm, 9.9 ± 0.5 mm, and 7. 4 ± 0.8 mm) comprise as many costal strips as there are longi- tudinal costae. While longitudinal costal strips attach to the middle of costal strips from the anterior ring, transverse costal strips from the mid-lorica costae span the width between neighbouring longitudinal costae. The reduction in diameter is occasioned by increasing overlaps between neighbouring transverse costal strips. The posterior transverse costa (diam. 3.1 ± 0.5 m m) comprises 4-5 costal strips. All transverse costae are internal relative to the longitudinal costae. The protoplast is located at the posterior end of the lorica chamber, within a suspensory membrane. Tectiform division assumed but not yet confirmed. Holotype: The specimen illustrated in Fig. 14a  This species was first observed by Leadbeater (1974;loc. cit. Pl. 2E/referred to as Parvicorbicula sp.) in samples from the Mediterranean (Split, Croatia and the Bay of Algiers). The single specimen illustrated is unmistakably morphome- trically identical to the material examined here (Table 1). The generic affiliation of this species was uncertain when first examined by Leadbeater (1974), and it has remained so ever since. It therefore seems appropriate at this stage, and while awaiting a molecular based unravelling of its phylogenetic connections, to at least formally describe this form, and as an interim solution allocate it to the 'pro forma' genus Coronoeca described here. The possibility initially indicated by Leadbeater (1974) of a certain resemblance between this form and species of Parvicorbicula is no longer valid considering the recent focus on regrouping selected species of Pleurasiga and core group members of Parvicorbicula species that are closely affiliated with the Parvicorbicula type species P. socialis (Leadbeater 2015, Thomsen et al. 2020a. Coronoeca tongiae is here ( Fig. 14a-g; Table 2) reported from the Andaman Sea, Thailand, West Australia, the eastern Mediterranean Sea, Alexandria, and the Gulf of California, Mexico.
Coronoeca patongiensis sp. nov. (Figs. 4g, 14h, i) Diagnosis: Lorica funnel-shaped (lorica height ca. 15 mm) and constructed of rod-shaped costal strips organized in ten longitudinal costae and three transverse costae. There are anterior spines corresponding in length to two thirds of an anterior longitudinal costal strip. A longitudinal costa is composed of four costal strips. Both the anterior transverse costa (diam. 12.5 mm) and the mid-lorica costa (diam. 8.0 mm) comprise as many costal strips as there are longitudinal costae, yet with a significantly larger overlap between neighbouring strips in the mid-lorica transverse costa. Transverse costal strips from both the anterior and the mid-lorica costa span the width between neighbouring longitudinal costae. The posterior transverse costa (diam. ca. 3 mm) comprises 4-5 costal strips. The anterior transverse costa is external relative to the longitudinal costae while the midlorica and the posterior transverse costae are internally located. The protoplast is located at the posterior end of the lorica chamber. Tectiform division presumed but not yet evidenced from the accumulation of costal strips in the collar region. Holotype: The specimen illustrated in Fig. 14i  While this species is superficially reminiscent of C. tongiae with reference to lorica shape, dimensions and costal strip configuration, there are, however, a number of distinguishing features, i.e. the pattern of junctions between transverse and longitudinal costal strips at the level of the anterior transverse costa (E-joins in C. patongiensis and T-joins in C. tongiae), and the placing of the anterior transverse costa (internal in C. tongiae and external in C. patongiensis). It is thus credible that the two species, once further studied using also molecular tools, cannot reside within the same genus. However, in an interim period it is convenient to be able to refer to a well-defined morphotype such as C. patongiensis using a formally established scientific name.
Coronoeca patongiensis has also been observed in samples from West Australia (Fig. 14h) and from the Sargasso Sea (Paul Davis; pers. com.).
Miscellaneous forms (Fig. 15) Under this heading we have assembled material of loricate choanoflagellate taxa that have either been observed too sparingly to allow for a formal species description, or unintentionally left out while dealing with the relevant genus in a previous paper (Stephanoeca urnula).
Choano sp. 1 (Fig. 15a) Lorica funnel shaped (lorica height: 10.1 mm) comprising eight longitudinal costae and two transverse costae (diam. 7.6 and 4.8 mm). The anterior tier of longitudinal costal strips project as spines. Costal strips from the anterior ring span the width between neighbouring longitudinal costal strips. This taxon resembles C. patongiensis but differs in having fewer transverse and longitudinal costae. Only observed (single specimen) in a water sample from the equatorial Pacific Ocean.
Choano sp. 2 (Fig. 15b) Lorica funnel shaped (lorica height: 14.8 mm) comprising ten longitudinal costae and two transverse costae (i.e. an anterior ring (diam. 12.0 mm) forming E-junctions with the longitudinal costae, and a posterior costa (diam. 4.6 mm) located at the joints between longitudinal costal strip number two and three counted from the anterior lorica end). This taxon, which is only observed once in a sample from the equatorial Pacific Ocean, resembles Cosmoeca phuketensis (see Thomsen and Østergaard 2019d) but differs because of a different number of longitudinal costae (there are consistently nine in C. phuketensis) and also by having a more complex posterior transverse costa. Choano sp. 3 (Fig. 15g) A single cell observed in a sample from the Andaman Sea, Thailand. The funnel-shaped lorica (lorica height: 19.5 mm) is constructed from eight longitudinal costae and two transverse costae. The anterior ring (diam. 12 mm) closes the lorica (E-junctions). The mid lorica transverse costa (diam. 9 mm) crosses posterior to the joints between the first and second longitudinal costal strips (counted from the anterior lorica end). Notice that the lon-gitudinal costal strips are exceptionally long (6-7 mm). The lorica chamber is pedicellate and posteriorly terminated by a basket-like configuration of obliquely oriented costal strips.
Stephanoeca urnula Thomsen, 1973 (Fig. 15c, d, h) This species was observed repeatedly in samples from the Eastern Mediterranean Sea outside Alexandria, Egypt. It is morphometrically (lorica height: 11 mm; diam. 3.7 mm (anterior opening), 7.2 mm (max. lorica width), and 4.5 mm (lorica waist)) in overall good agreement with the Danish type material (Thomsen 1973), and also material sampled from the innermost parts of the Baltic Sea (Thomsen, 1979, Vørs 1992, Ikävalko and Thomsen 1997, Ikävalko 1998. Stephanoeca urnula has additionally been recorded from Lake Saroma, Japan (Takahashi 1981) and the South Atlantic coastal zone, Brazil (Bergesh et al. 2008;14-15 PSU). The majority of samples yielding S. urnula are from low-saline regions such as the Baltic Sea. The occurrence of this form in an eastern Mediterranean high salinity environment is thus surprising. The exact habitat sampled is perhaps impacted by the river Nile freshwater outflow creating brackish water niches that may allow S. urnula to thrive, or is it rather that the Egyptian material, despite an overall similarity, does represent a separate taxon with markedly different habitat demands. It is in this connection relevant to refer to recently described nudiform Stephanoeca-like species (Enibas tolerabilis and E. thessalia; Schiwitza et al. 2019;Schiwitza and Nitsche 2021) that are morphologically reminiscent of S. urnula yet with a significantly different autecological signature. Based on laboratory tests is was found (Schiwitza et al. 2019) that E. tolerabilis is able to survive within a salinity range of 0-70 PSU.
Savillea sp. (Fig. 15e, f) Two cells observed in samples from the Sargasso Sea. They differ from Savillea parva (Ellis, 1929) Loeblich III, 1967, by having a significantly more open-meshed pattern of spiralling and longitudinal costae. The lorica is 13-15 mm high, and the diameter at the orifice 4-5 mm and in the mid-lorica region 8-9 mm. Similar material has previously been observed in Sargasso Sea samples (Paul. G. Davis, pers. com.; lorica height 14.2 mm, diam. at orifice 4.2 mm, max. diam. 7.3 mm).

Species diversity
The total number of species encountered in our warm water samples is 80 (Table 2). This represents close to 50% of all loricate choanoflagellate species described so far. There is a fairly obvious link between the material available to us, in terms of the mere number of water samples  Thomsen 1979, Vørs 1992, Ikävalko and Thomsen 1997, Ikävalko 1998 Thomsen,unpublished;(19) Weddell Sea, Antarctica; Thomsen et al. 1990(20) Prydz Bay, Antarctica; Marchant 1985, Marchant andPerrin 1990;  and LM/TEM preparations, and the total number of species listed from each individual site. The most thoroughly screened locations (West Australia and the Andaman Sea) are each represented by more than 60 species, whereas for the least intensively sampled regions (the Caribbean Sea, and the Eastern Mediterranean Sea) we have only listed approximately 30 species. For comparison it can be emphasized that similarly intensely sampled regions, i.e. Danish coastal waters and Disko Bay, West Greenland, are each represented by a maximum of 45 species. It thus appears that the tropical Indian Ocean sites sampled (the Andaman Sea and West Australia) do represent unique biodiversity hot spots.
To put the regions here analysed into a proper perspective, we have assembled biodiversity data worldwide (Fig. 16A, B) and conducted a matrix analysis (Fig. 17) in the shape of a two-way clustering of localities (35) versus species (presence-absence data; the diagram includes the 50 most decisive species). It is immediately obvious (Fig. 17), judged from the overall diagonal patterning of the matrix, that specific subsets of samples largely defined by temperature are matched by fairly clear-cut subgroups of species. The warm water sites reported on here cluster closely together (ca. 60% similarity) with maximum similarity (ca. 90%) between the Andaman Sea and the West Australian collections, and match up with a cluster of 9 species that have their maximum occurrences in such warm water habitats (Fig. 17D/  H; i.e. Campyloacantha imbricata, Campyloacantha spinifera, Cosmoeca ceratophora, Cosmoeca subulata, Pleurasiga echinocostata, Polyfibula elatensis, Stephanacantha dichotoma, Syndetophyllum pulchellum, Thomsenella infundibuliformis). It is similarly evident that there is a well-defined square within the matrix that defines associations between both Arctic and Antarctic cold-water habitats and cold-water adapted species (Fig. 17B/F; i.e. Acanthocorbis unguiculata, Bicosta antennigera, B. spinifera, Calliacantha longicaudata, C. natans, Diaphanoeca pedicellata, Parvicorbicula quadricostata, P. socialis). Notice that there is a North Atlantic cluster of sites (Baltic Sea, Denmark, Southampton (UK) and Rhode Island (USA)) that is overall very similar to the cold-water community, however, with a wider range of species represented. The matrix segment marked 'C' (Fig. 17) comprises species that are truly cosmopolitan forms (i.e. Bicosta minor, Cosmoeca norvegica, C. ventricosa, Calliacantha simplex, Diaphanoeca grandis, Parvicorbicula circularis, Pleurasiga minima). The most divergent matrix element (Fig. 17A/E) is represented by Antarctic Weddell Sea icebiota samples which display a wide range of forms not encountered elsewhere. Samples from South Brazil and the North Pacific Central Gyre cluster separately very likely due to limitations in the number of taxa recorded from these sites (Fig. 16B). The inevitable conclusion to be extracted from the kind of analysis undertaken here, is that there are consistent and well-defined temperature dependent species associations with clear distributional constrictions, as well as groups of cosmopolitan species that have the potential to occur everywhere.

Conclusions
While the genus Polyfibula is unambiguously defined based on overall lorica constructional details and especially the facets on transverse costal strips, it applies to all remaining species examined here, that the generic allocation is negotiable and likely to change once supplemental molecular data becomes available. In order to generally move loricate choanoflagellate taxonomy ahead, it is urgently required to seek supporting molecular evidence. A cultivation approach is one obvious way to go (see e.g. Nitsche and Arndt 2008;Schiwitza et al. 2019). However, it is likely that a vast range of species, and in particular those that prefer more oceanic habitats, will be very hard and time con-suming to ever establish in culture. An alternative, or perhaps rather supplemental approach, is sequencing based on the extraction of single cells from a suspension of nanoflagellates concentrated from freshly collected water samples (see e.g. Nitsche et al. 2017;Schiwitza and Nitsche 2021). While this does work adequately when dealing with large forms, that can be recognized and unambiguously identified from mixed populations at low magnification in the inverted microscope (Â40-60 objective), this approach will not suffice, when it comes to small loricate forms that share basic features in lorica design and overall construction. What is needed to move fast ahead, linking specific morphotypes with their unique sequences, is an approach that allows for an initial incontestable species identification, based on e.g. an examination of natural, wet and non-coated material in an environmental scanning electron microscope, and a subsequent extraction of the specific cell for sequencing. While the identification part and the position-fixing is fairly straightforward to accomplish, it is the subsequent manipulation (extraction) of the cell that is likely to be seriously challenging. No matter the approach taken it appears that an initial accessibility to flow-sorted cells, prepared as described by e.g. Kamennaya et al. (2018), might be essential in optimizing the success-rate in terms of securing quality approved data linking specific morphotypes to molecular profiles.
It is evident from Fig. 16A that the loricate choanoflagellate research community has by now achieved, based on classical light and electron microscopical descriptive techniques, a fairly comprehensive global coverage of species distributions. This has in turn contributed to e.g. a basic understanding of how temperature in particular impacts on large-scale species occurrence patterns. The approach that we have taken in the current series of papers on warm water loricate choanoflagellate biodiversity, is to reduce often complex regional sampling programmes (along multiple transects covering several depths) to a single species file. While this has proved adequate with reference to the taxonomic and morphometric data analysis, and also in the context of underpinning that temperature is a decisive factor in global biodiversity patterns, it conceals, however, species associations differentiating e.g. near coastal versus offshore habitats, surface water versus fluorescence maximum layers, and also where relevant (e.g. the Andaman Sea, Thailand) possible seasonal patterns in species occurrences. Once adequately progressed the molecular tools will be keystone elements in puzzling out any such small-scale patterns in species occurrences.

Author contribution statement
Helge A. Thomsen (HAT) has conceived and designed the analysis, generated funding, and collected and processed samples. HAT is further responsible for compiling and writing the paper. Jette B. Østergaard (JBO) has been much involved in data collection and sample processing with reference to the Andaman Sea and the Pacific Ocean. Seiko Hara (SH) has contributed data from the Seto Inland Sea, Japan, and has been instrumental in finalizing the later versions of the manuscript.