Based on two Andean Altiplano samples and on light and scanning electron microscopy analyses, we present six new species of “araphid” diatoms in the genus Pseudostaurosira, P. aedes sp. nov., P. frankenae sp. nov., P. heteropolaris sp. nov., P. oblonga sp. nov., P. occulta sp. nov., and P. pulchra sp. nov. Additional data are provided for four other known taxa, Nanofrustulum cataractarum, N. rarissimum, P. sajamaensis and P. vulpina, the latter species corresponding to a stat. nov. based on a variety of P. laucensis. Each taxon is described morphologically and compared with closely related published taxa, using characters such as axial area, virgae, vimines, areolar shape, volae, internal striae depositions, spines, flaps and apical pore fields, which are not usually used for species distinction within the genus. It is our intention that the detailed morphological descriptions of each taxon and the elaborate comparative tables we provide serve as a basis for correction of neo and paleo-databases for the Altiplano to produce a better account of autecological data and ecological change in the region. Some arguments for our continued use of a morphologically based approach are given in the context of rapid environmental degradation in the Andes and the difficulties in applying molecular approaches in countries such as Bolivia.

Andean mountains, fragilarioid diatoms, morphology, South America, traditional taxonomy

In the last two decades, many new “araphid” taxa have been described, clarifying the morphological concepts of existing genera or better delimiting the boundaries of widely reported species (e.g. Lange-Bertalot and Ulrich 2014; Wetzel and Ector 2015; Wengrat et al. 2016; Almeida et al. 2017; García et al. 2017; Van de Vijver et al. 2020a). The study of type material helped in the latter endeavor, which coupled with illustrated reports and newly found populations, gave a clearer view of diagnostic characters and added other features that had not been used before for recognition of purportedly well-known species (e.g. Edlund et al. 2006; Cejudo-Figueiras et al. 2011; Wetzel et al. 2013a, b; Talgatti et al. 2014; Delgado et al. 2015; Van de Vijver et al. 2020a, b). Such is the case, for example, with Pseudostaurosira brevistriata (Grunow) D.M. Williams & Round (Morales et al. 2015, 2019b) and Staurosirella pinnata (Ehrenberg) D.M. Williams & Round (Morales et al. 2013a, 2019a). The literature for both is extensive, revealing a history of taxonomic drift, lumping and imprecise reports of their autecology (Morales et al. 2013a, 2014c, 2019b).

These morphological studies continue to be important in the resolution of taxonomic issues and taxa delimitation. New morphological descriptions and taxonomic revisionary work provide a series of testable hypotheses that constitute the grounds upon which further progress can be made in fields such as systematics, ecology, conservation, etc. (de Carvalho et al. 2007; Haszprunar 2011).

Though molecular studies are becoming increasingly important in the resolution of taxonomical issues at the species level, both sources of information, morphological and molecular, ought not to be divorced and are rather complementary since morphological studies generate hypotheses based on the phenotype, while molecular studies do it based on the genotype. One dataset can be used as a confirmation of the other. The concatenation of both sources of information could produce a stronger and better-supported taxonomic system that can be translated, for example, into a practical tool to be used at the bench during routine identification analyses (Kahlert et al. 2019).

However, the colossal task that represents the production of fully operating barcode datasets (Zimmermann et al. 2014; Kelly et al. 2018) that are applicable to nature deems “traditional” morphological analyses a continued fast and viable way to produce data and hypotheses on taxa identities and distinctiveness. The same can be said for reliable phylogenies that aim to express natural classifications (see Li et al. 2018 and discussion in Morales et al. 2019b) but production of phylogenies is another matter, a different stage in the process of studying biodiversity that we are not concerned with in the present contribution. Here we deal only with a first stage of discovery, description of traits and a comparative analysis to justify the hypothetical placement of the treated taxa under given genera.

The study of “araphid” diatoms from high Andean ecosystems is important since they are frequent in current and paleoecological samples. Their abundance and distribution have been used to determine past climate, water level and precipitation changes, salinity and ionic composition, and temperature variations (e.g. Servant-Vildary 1978; Servant-Vildary and Roux 1990; Fritz et al. 2004; Tapia et al. 2006). But also from the taxonomic standpoint, it becomes relevant to describe species and produce inventories and autecological data for Andean “araphids” and other diatoms, especially because mountainous areas are affected more rapidly by climate change than any other land ecosystems (Marengo et al. 2011; Cuesta et al. 2012; Michelutti et al. 2015). Coupled with land use effects, threats to ecological stability with the anticipated negative effects on human and other populations are beginning to be observed in these areas (Suárez et al. 2011). A thorough knowledge of the taxonomy and autecology of diatoms could provide an aid in the conservation of Andean areas and their communities.

The examination of recent samples from Bolivia and Argentina has shown that the Andes contains hot spots for “araphid” species diversity (e.g. Morales et al. 2012b, 2019b; Grana et al. 2018; Seeligmann et al. 2018; Guerrero et al. 2019). Study of these sites could facilitate the taxonomic clarification of several known taxa, quickly produce new species and generate inventories that can then be applied to other Andean regions.

The present paper aims to continue the morphological description of diatom taxa present in the region contiguous to Lake Titicaca, concretely in the Desaguadero River and adjacent zones. This area is affected by natural soil erosion, typical of the Bolivian Altiplano, but also by land use and water use changes that have been affecting the area for several decades (UNEP-United Nations Environmental Program 1996). The recent international news about the drying of neighboring Lake Poopó, the site where a substantial amount of fauna and flora thrived and from where human groups, descendants of the millenary Urus tribes, have been displaced (Richard and Contreras 2015), furnished evidence of the urgency of basic studies such as the present one. The long-term goal is to improve the paleolimnological/paleoclimatic characterizations conducted in the area (e.g. Servant-Vildary 1978; Servant-Vildary and Blanco 1984; Tapia et al. 2003) and to provide baseline data for conservation and management practices in the region.

Concretely, we present six new species together with comparative analyses with published morphologically closely related taxa, and additional morphological information and comparative data for other four species described from the Andes and elsewhere. For all ten taxa, a pertinent discussion is presented to aid in their distinction and identification.

The study area, the southern region contiguous to Lake Titicaca, was already described in a geographical and ecological context by Morales et al. (2012b, p. 42–44) and Grana et al. (2015, table 1, fig. 4 and p. 815). Epipsammon material used in the present study is the same as that described in Grana et al. (2015), collected from rivers Desaguadero and Sajama with the aid of a turkey baster and fixed with 20 drops of 40% formaldehyde in the field.

For LM analysis, subsamples of 20–30 mL were mixed with a similar volume of 70% HCl. The mixture was boiled for 45 min and rinsed 8 times using distilled H₂O. Drops of cleaned slurry were dried on coverslips overnight at room temperature. Permanent slides were mounted using the synthetic medium Naphrax. Slides were analyzed using a Zeiss Universal microscope equipped with differential interference contrast optics, a 1.25 optivar, and a Plan 100X, 1.25 NA, immersion objective. Images were taken using a Jenoptik CF color digital camera and ProGres CapturePro ver. 2.8 software.

For SEM analysis, about 10 to 20 mL aliquots of raw samples were digested with concentrated H₂O₂ and heated for 24 h using a sand bath. Then, samples were allowed to cool and settle (ca. 1 cm/h) and 80 to 90% of supernatant was eliminated by vacuum aspiration. A volume of 1 mL of HCl acid (37%) was added and the preparation was allowed to settle for 2 h. Subsequently, the sample was rinsed and decanted three times using deionized water. Approximately 100 mL aliquots of clean material were filtered and rinsed with deionized water through glass fiber filters with a 3 μm pore diameter. Coating with platinum was accomplished using a BAL-TEC MED 020 Modular High Vacuum Coating System for 30 s at 100 mA. A Hitachi SU-70 electron microscope operated at 5 kV and 10 mm distance was used for SEM analysis. Micrographs were digitally manipulated and plates containing LM and SEM pictures were mounted using Photoshop CS3.

Identification of taxa was performed using literature published for South America (Metzeltin and Lange-Bertalot 1998, 2007; Rumrich et al. 2000; Metzeltin et al. 2005). Taxonomic articles by Frenguelli (1939), Patrick (1961), Manguin (1964), Hohn (1966), Servant-Vildary (1986), Morales and Vis (2007) and Morales et al. (2007) were also used. Additionally, non-South American references were used such as the general floras of Patrick and Reimer (1966, 1975), Krammer and Lange-Bertalot (1986, 1988, 1991a, b), Simonsen (1987), Lange-Bertalot (1993), Hofmann et al. (2011), and Lange-Bertalot et al. (2017), as well as references specialized in certain genera such as Navicula (Lange-Bertalot 2001), Pinnularia (Krammer 2000) and cymbelloids (Krammer 1997a, b). Descriptions of the new taxa are based on measurements of 30 valves and observation of more than 100 individuals of each taxon under both LM and SEM. Morphological terminology follows Anonymous (1975), Ross et al. (1979) [both references used for terminology applied to striae, areolae and spines], Barber and Haworth (1981) [for terminology related to valve shape and striae orientation], Williams and Round (1987) and Round et al. (1990) [both references used for terminology on areolar substructures, girdle band features and apical pore field characteristics].

Nanofrustulum cataractarum, as seen in samples from the Desaguadero and Sajama rivers, is very similar to the type and other populations reported from around the world (Wetzel et al. 2013a; Grana et al. 2015; Beauger et al. 2019; Genkal 2021). The smaller dimensions of Bolivian specimens only expanded the initial measurements given by Hustedt (1938).

The more noticeable blisters on the mantle for Bolivian specimens could be due to the state of preservation of the material (more recent collection from Bolivia) and the possible higher availability of silica in the environment.

The lack of apical pore fields, stipules and flaps are typical in this taxon, but the most noticeable characteristic at the time of its identification under LM is the round shape of its valves and areolation pattern of the valve face and mantle, which resemble the smallest members of Aulacoseira Thwaites.

Nanofrustulum rarissimum, as discussed by Morales et al. (2019b), belongs in Nanofrustulum due to the quasifract nature of its girdle elements. The valvocopula in this case, however, is entire and ligulate. This difference with N. cataractarum that has all girdle elements quasifract has not been assessed in detail (Morales et al. 2019b), especially regarding the consequences for classification at the genus and species levels. It is known that other species can form morphologically different girdle elements (see the case of Nitzschia transtagensis E. Morales, Novais, C.E. Wetzel, Morais & Ector in Morales et al. [2020, p. 34, figs 2–26], and other examples therein). A more detailed study of the variation of this character within the species currently assigned to Nanofrustulum is required.

Pseudostaurosira sajamaensis has large areolae proportional to its size (Morales et al. 2012b), which together with the gradual valve face/mantle transition, the sagittate-profiled spines with single or diapason-shaped tips, bearing serrate borders pointing downward are the main features to look for at the SEM level (Morales et al. 2012b, Fig. 2E, F, Table 1). Also characteristic at the latter level is the infrequent presence of a V-shaped cleft in the posterior side of the spine body. In the context of the taxa contrasted in Table 1, these are the features that are unique to this taxon.

Pseudostaurosira sajamaensis is found in the same Desaguadero River sample together with P. aedes sp. nov. and P. pulchra sp. nov. However, at the LM level, the elliptic valves with pointy ends, the proportionately larger areolae, and the gradual transition between valve face and mantle in P. sajamaensis readily differentiate this taxon from the other two. The SEM features mentioned above, and which are defining for this taxon, can also be used to distinguish it from P. altiplanensis at this level (Lange-Bertalot & Rumrich) E. Morales (Rumrich et al. 2000, p. 220, pl. 14, figs 1–8; García et al. 2017, p. 112) (Table 1).

Pseudostaurosira linearis (Pantocsek) E. Morales, Buczkó & Ector (in Morales et al. 2019b, p. 276, figs 3, 4) is another taxon with a gradual valve face/mantle transition, but it has very different features to those taxa included in Table 1. This taxon is fossil, and tends to produce longer valves; the axial area is at the same level as virgae in external and internal views, has spines with T-shaped tips and well-developed M or V-shaped stipules, and the apical pore fields are more- or less-developed with cavernous appearance externally and opening internally into a non-depressed area.

Pseudostaurosira sajamaensis has been recorded at the LM level from the Tunari Cordillera, in the Department of Cochabamba (E. Morales pers. obs.) located more than 200 km to the east of the Sajama and Desaguadero rivers. This cordillera is part of a long range that branches off the main Andes mountains, penetrating Bolivian territory and receiving the name of Eastern Cordillera (see “Study area” description in Morales 2020). These records, however, need to be confirmed with SEM. If confirmed, the range of this diatom would be extended to the eastern limits of the Bolivian Altiplano and their confluence with the Bolivian Dry Valleys.

Pseudostaurosira pulchra sp. nov. has the main distinguishing feature of Pseudostaurosira, the short and wide vimines (Morales et al. 2019b). Because the striae are mostly composed of two areolae, the vimines are mostly restricted to the valve face/mantle junction. The characteristics of the spines interrupting the striae, the areolae and associated structures (volae and flaps), the blister-like depositions and girdle elements are all in accordance with species currently ascribed to this genus.

Table 1 shows that the diagnostic features of P. pulchra sp. nov. are the narrow lanceolate valves with rostrate apices in larger specimens, becoming broadly rounded in smaller ones. Also the virgae wider than the striae, that are raised with respect to the axial area in internal and external views are typical in this species. Finally, the spines having a base that is wider than the vimines they sit on, and a body that is flattened with concave margins and has a flat top, or small bifurcate projections are also diagnostic in this taxon.

The taxon with the most similar morphology to P. pulchra sp. nov. is Pseudostaurosiropsis geocollegarum (Witkowski & Lange-Bertalot) E. Morales (Witkowski et al. 1995, p. 734, figs 16–22; Morales 2002, p. 104, pl. 1, figs 1–9) (Table 1). At the LM level both have lanceolate valves, but in P. pulchra sp. nov. they are narrower (2.4–3.0 vs. 2–4 µm in P. geocollegarum), and the ends vary from rostrate to broadly rounded (subrostrate in P. geocollegarum). At the SEM level the most conspicuous difference is that P. geocollegarum does not possess volae and has rotae, a structure completely lacking in all Pseudostaurosira. Pseudostaurosiropsis geocollegarum also has spines, the body of which starts with a pyramidal shape, becoming cylindrical toward the top, bearing bifurcate ends, a defining feature for the species (Table 1). Additionally, the apical pore fields in P. geocollegarum have a cavernous appearance with few pores that open as large isolated pores at the valve interior; this is also a defining feature for the species.

Pseudostaurosira pulchra sp. nov. has not been observed in other samples from the Altiplano and seems to be restricted to the Desaguadero and Sajama regions.

Pseudostaurosira aedes sp. nov. has short and wide vimines, which place it in Pseudostaurosira. The vimines are mostly restricted to the valve face/mantle junction since the striae are commonly composed of only two areolae, one on the valve face and the other, usually larger, on the valve mantle. The other features such as spines located along the striae, the areolae and subareolar structures (volae and flaps), the blisters and girdle elements, are all in accordance with species currently placed in Pseudostaurosira.

Despite the apparent difficulty in the distinction of this diatom from other similar taxa (Table 1), it has distinctive features that set it apart from them. The narrowly elliptic shape with rounded ends, the arrowhead-like spines (configuration given by the well-developed stipules) with a conical body and serrate tips, and the apical pore fields composed of only a few round poroids and opening internally into a single linear depression are all diagnostic features of this species.

At the LM level, the most similar taxon to P. aedes sp. nov. is Pseudostaurosiropsis connecticutensis E. Morales (2001, p. 117, figs 7a-l) (Table 1). Especially for smaller specimens, both present elliptic valves but the stria density is much higher in P. connecticutensis (17–20 vs. 15). Also, the spines in the latter are flattened with a biconcave-spatulate body and bifurcate ends. As a member of Pseudostaurosiropsis, P. connecticutensis has rotae and lacks volae. Additionally, this taxon has virgae that are at the same level of the axial area in internal view, while they are slightly raised in internal view. Spines with a flattened, spatulate body, concave on the sides, and with bifurcate ends. These characteristics of the virgae with respect to the axial area, and the spines are defining features for P. connecticutensis.

Smaller representatives of P. aedes sp. nov. can also resemble Pseudostaurosira altiplanensis (Lange-Bertalot & Rumrich) E. Morales. At the LM level, however, P. altiplanensis is much wider (2.8–3.6 vs. 2.1–2.6 in P. aedes sp. nov.), the valves are broadly elliptic instead of narrowly elliptic with rounded ends and the striae are long, composed of transapically very elongate areolae. At the SEM level, the virgae are wide and slightly raised together with axial area in both external and internal views; the spines are narrower than vimines, with a cylindrical body with straight sides, spatulate tips with small and thin lateral projections, bearing little-developed stipules. The blisters on the abvalvar side of the mantle are comparatively smaller. All these SEM features are defining characters for P. altiplanensis (Table 1).

Pseudostaurosira aedes sp. nov. was only found in the Desaguadero River in the present study, but it was illustrated before by Rumrich et al. (2000, pl. 13, fig. 26, only figure that is not numbered in the plate) and identified as “Staurosira brevistriata Grunow” (Pseudostaurosira brevistriata (Grunow) D.M. Williams & Round, 1987, p. 276; for figures of type material refer to Morales et al. 2015, figs 107–143 since figures in Williams & Round and incorrectly labeled). The valve shape, the features of the areolae, spines and blisters are all confluent with what is described here as P. aedes sp. nov.

Pseudostaurosira ushkaniensis Kulikovskiy & Lange-Bertalot (in Kulikovskiy et al. 2015, p. 28, pl. 16, figs 1–12, pl. 17, figs 1–8) resembles both P. pulchra sp. nov. and P. aedes sp. nov. under LM. However, the mantle areolae in P. ushkanenis, being large and occupying almost the entire shallow, curved mantle in a pervalvar direction, are clearly visible showing two rows of these structures per stria in valve view. At the SEM level, the spines are located on virgae, have a cylindrical body and a spatulate tip. The apical pore fields are well-developed with neatly arranged rows of poroids in external view and a depressed elliptic plate with clearly round poroids in internal view. All of these features differ from the two Bolivian taxa (see them in Table 1).

Pseudostaurosira heteropolaris sp. nov. has wide and short vimines, a character that places it in Pseudostaurosira. This species is distinguished by its short, ovoid to elliptic, heteropolar valves, the wide base of the volae which give the areolae a C-shape appearance, the pinnatifid profuse bifurcations of the spine tips and the small blisters on the abvalvar edge of the mantle (Table 2).

The evident heteropolar configuration of P. heteropolaris sp. nov. is shared with P. clavatum E. Morales (2002, p. 107, pl. 1, figs. 22–34, pl. 4, figs 1–6), but this is a very different species with larger valves (8–20 µm), coarser striae (11–12), wide, elliptic to trapezoid areolae on valve face and mantle (one on each), profusely bifurcate volae, flat, hollow spines with serrated borders and two ligulae projected laterally, and well-developed apical pore fields on both valve apices.

Another species with evident heteropolar shape is P. conus Kulikovskiy & Lange-Bertalot (in Kulikovskiy et al. 2015, p. 26, pl. 15, figs 15–18). At the LM level the valves are clavate with rostrate, sometimes subcapitate head pole and a very fine foot pole. External views of this taxon under SEM are unknown, but from the single figure presented by Kulikovskiy et al. 2015 it can be seen that the spines are small and conical and that the apical pore fields are well-developed and composed of several rows of poroids, features absent in P. heteropolaris sp. nov.

The same authors presented P. gomphonematoidea Kulikovskiy & Lange-Bertalot (in Kulikovskiy et al. 2015, p. 26, pl. 19, figs 1–3), another clavate taxon with a broadly rounded head pole and a thinner foot pole. The areolae are round to elliptical and the volae are not as developed as in P. heteropolaris sp. nov. The spines are completely flat and the apical pore fields are well-developed, features not present in the new taxon from Bolivia.

As evident in Table 2, P. heteropolaris sp. nov. is different from morphologically similar species in the genus, from which it can be distinguished in a first instance under LM by the features cited above, especially by the small size and heteropolar valves. The rest of the diagnostic features need to be revealed by SEM. Among the latter, the features of the areolae and spines completely separate this taxon from the others in the present manuscript.

Pseudostaurosira vulpina stat. nov. has a triradiate form, the first key feature to its identification under LM. This combined with the swellings mid-way between arms most surely give a positive identification. Confirmation at the SEM level is given by the apical pore fields, somewhat depressed into the three valve apices and opening to the valve interior by a single non-depressed porous plate. In Table 3, we also annotate that the externally depressed axial area with respect to the virgae, and internally at the same level as the latter is an exclusive feature of P. vulpina among triradiate forms. Within the latter, P. vulpina is also the only one possessing small conical spines.

Depositions in the internal surface of the striae have also been reported in Pseudostaurosira decipiens E. Morales, G. Chávez & Ector (in Morales et al. 2012b, p. 44, figs 2–10, 39–44). However in P. decipiens there are two superimposed disks on each areolar opening, while in P. vulpina there is only one, inverted cone-like structure, hollow at the center or with extra siliceous deposition in its interior (Fig. 7F). These cones seem to appear as material accumulates over the copiously branched volae. Since the areolae are elongated in P. vulpina, the base of the cones is distorted, assuming a somewhat triangular or ovoid configuration (Fig. 7F).

P. vulpina as presented in Rumrich et al. (2000) lacks the internal cone-like depositions, but this seems to be due to erosion on the valves since the volae seem to have also been lost to a great extent from the areolae. These cone-like depositions are also found in the recently described fossil Pseudostaurosira crateri Marquardt & C.E. Wetzel (in Marquardt et al. 2021, p. 107, figs 1–57). However, this latter taxon is very different from the triradiate P. vulpina in that it has a lanceolate shape, very narrow striae with more areolae on the valve mantle than on the valve face, which results in a wide axial area, depressed externally and internally, and apical pore fields in internal view reminiscent of P. parasitica (W. Smith) E. Morales (2003a, p. 287, figs 27–43, 54–54, 60, 64), i.e. an elevated plate that contains several round poroids. Externally, these apical pore fields resemble those in P. vulpina, although they are much larger proportionately to the valve size in P. crateri.

Pseudostaurosira iztaccihuatlii V.H. Salinas & D. Mora (in Salinas et al. 2020, p. 196, figs 18–35) is most probably conspecific with P. vulpina. The only two differences between the population from Mexico and the Andean population are the larger spines interrupting the striae of the former and the shape of the internal striae depositions (Table 3, features that have not been traditionally used for separation of species within the genus). The structure of the areolae and volae structure is similar in both taxa and there is no external or internal velum, as misinterpreted by Salinas et al. (2020); if a velum were present, P. iztaccihuatlii would have to be placed in a different genus. Both the structure of the areolae and volae are extensively used to separate Pseudostaurosira from other genera and to separate its species, as we have done herein. The internal striae depositions are ring-like in the latter and cone-like in P. vulpina.

Also from Table 3, it is clear that Pseudostaurosiropsis triradiatum (E. Morales) Kulikovskiy, Glushchenko & B. Karthick (Morales 2005, p. 129, Figs 74–79, 127–132; in Radhakrishnan et al. 2020, p. 173) is different from P. vulpina due to its axial area in the shape of a triangle with concave sides, the much wider virgae than striae, the circular areolae with rotae, small conical spines wider than vimines, and the apical pore fields that are not depressed exteriorly, but they do internally.

A third taxon that could be compared with P. vulpina is Staurosira mercedes Lange-Bertalot & Rumrich (in Rumrich et al. 2000, p. 224, pl. 10, figs 12–14), a taxon that had been introduced under the name Fragilaria brevistriata var. trigona Lange-Bertalot nom. inval. without a diagnosis in Krammer & Lange-Bertalot (1991a, pl. 117, fig. 7B). No SEM images of S. mercedes have been published, but at the LM level the valves are triangular with concave sides (boomerang-like) and the ends are cuneate rather than rostrate as they are in P. vulpina. Lange-Bertalot also introduced the name Staurosira pseudoconstruens var. trigona (in Rumrich et al. 2000, pl. 15, figs 1, 2), but this name was not accompanied by a diagnosis either. The LM figure the authors presented (fig. 2) seems to be a teratological form and has a similar general appearance in the lower side of the triradiate shape as a medium-sized valve of P. vulpina. The SEM image presented in Rumrich et al. (2000, pl. 15, fig. 1) confirms this. The shape of the areolae, the position of spines and the sunken apical pore fields in the close-up image are all similar to P. vulpina. We also note that Metzeltin & Lange-Bertalot (1998, pl. 2, fig. 5) presented a valve of P. vulpina (judging by valve shape and features of striae, areolae, spines and apical pore fields) that they identified as “Fragilaria brevistriata (Grunow s. lato) var. trigona Lange-Bertalot” a name that appears only in this text and without a formal description.

The change in status of P. vulpina is here justified by the finding of a population with mixed frustule sizes, a probable indication that the species is reproducing asexually and sexually, independently from the nominate variety, P. laucensis, and growing isolated from it in the Desaguadero River.

This taxon has been reported and illustrated from the Argentinian (Tchilinguirian et al. 2018), Bolivian (Morales et al. 2007) and Chilean Altiplano (Rumrich et al. 2000), with possible records from Europe still to be confirmed by SEM. It is possible that it was also identified with other names. For example, Servant-Vildary (1978, p. 3, pl. 2, fig. 13, see also LM image in Servant-Vildary 1986, pl. 2, fig. 27) presented a drawing that closely resembles our Figs 6C and D, with triangular shape and inflated sides and mammillate ends, which the author named “Fragilaria construens (Ehr) Grun var. exigua (W. Smith) Schuls[sic]” (=Staurosira construens var. exigua (Ehrenberg) H. Kobayasi (in Mayama et al. 2002, p. 90, for illustrations refer to Krammer and Lange-Bertalot 1991a, pl. 117, figs 4–7 (LM) and Potapova 2014, p. 80, fig. 98 (SEM, under “Staurosira construens f. exigua”)). From the latter references the var. exigua is distinguished by subcapitate ends, incipient spines developing on virgae, and the unbroken striation pattern, since the striae are formed by small, apically elongate areolae. Additionally, the apical pore fields are not sunken and contain several rows of neatly arranged poroids.

Pseudostaurosira frankenae sp nov. is an additional cruciform species included in the genus (Table 4). It has the main feature of species currently assigned to it, namely the wide and short vimines, but it also shares with them the features of the areolae, spines and flaps, apical pore fields and girdle elements.

This new species has several distinguishing features that set it apart from other congeneric taxa with cruciform valve outline. The virgae are slender than striae, internally the striae bear a single elliptic, occluding disk (a character unique in the entire genus), Spines have a triangular basal configuration when viewed from the side; the areolae bear persistent flaps (Table 4). Additionally, the apical pore fields are covered by a siliceous deposition that only reveals a single row of poroids. We have not studied variation of this latter feature, it seems to be constant in the species but it requires confirmation.

Due to their cruciform shape, P. australopatagonica M.L. García, L.A. Villacís, Maidana & E. Morales (in García et al. 2021, p. 3, figs 2–35), P. caballeroae V.H. Salinas, D. Mora, R. Jahn & N. Abarca (2020, p. 199, figs 36–50) and P. pseudoconstruens (Marciniak) D.M. Williams & Round (1987, p. 277, figs 28–31 mistakenly under “Pseudostaurosira brevistriata”) are the closest morphological relatives to P. frankenae sp. nov. However, from Table 4, it can be seen that P. australopatagonica has the volae forming an internal dendritic pattern, which is a character unique for the species. Additionally, this latter species has incipient spines forming arched, convex structures on the vimines, interrupting the striae at the valve face/mantle junction. The species from Argentina lacks stipules as P. frankenae sp. nov. does, but the latter has flaps, which are absent in P. australopatagonica.

In the case of P. caballeroae, the spines are thin and flat, forming an undulate dentate pattern over vimines (where most of the spine base and lower body lie) and virgae. While this species and P. frankenae sp. nov. lack stipules, the latter has flaps, which are lacking in P. caballeroae.

Pseudostaurosira pseudoconstruens has the closest overall valve shape to P. frankenae sp. nov. However, P. pseudoconstruens has the spines on the virgae and not on vimines as the rest of the species discussed in the present work (Williams and Round 1987). The base of those spines is shorter and they possess highly branched tips. Both stipules and flaps are lacking in this species. Additionally, the apical pore fields are highly reduced and are not cavernous as in P. frankenae sp. nov.

Pseudostaurosira decipiens E. Morales, G. Chávez & Ector (in Morales et al. 2012b, p. 44, figs 2–11, 39–44), P. laucensis and P. parasitica (W. Smith) E. Morales (2003a, p. 287, figs 27–43, 54–58, 60, 64) have a lanceolate valve shape, this being the first distinguishing feature to separate them from P. frankenae sp. nov.

From Table 4, it is worth noticing that P. laucensis, a taxon that can be distinguished from morphological related species by its incipient spines occurring on virgae and vimines along the entire valve face/mantle transition, its internal ring-like depositions on the striae and its non-cavernous apical pore fields, had already been shown by Servant-Vildary (1986, pl. 3, figs 31b–36), identified as “Fragilaria brevistriata Grunow”. Both references show populations with the same valve features, although the latter reference shows a valve interior with the typical ring-like depositions on the striae. These depositions are unique to P. laucensis among taxa with cruciate to lanceolate valves. The ring-like configuration, however, is shared with the triradiate P. iztaccihuatlii (see Table 3 and discussion above). These two taxa also share the apical pore field configuration and the difference in areolar width between valve face and valve mantle areolae.

Regarding the incipient spines in P. laucensis, these also occur in P. vulpina as shown in Fig. 7E, but also in Rumrich et al. (2000, pl. 10, figs 8, 10, 11). Both taxa also share the apical pore field configuration.

Pseudostaurosira occulta sp. nov. is distinguished from similar species under LM by its lanceolate shape with subrostrate, somewhat square and broadly rounded apices (Table 5). In this latter table it can be seen that there are several distinguishing features typical of this new species at the SEM level. From these, the circular depression on the spine body, subtended by an incipient stipule, stands out. Also, the flap coverings, twisted, almost externally completely obstructing the apical pore fields are characteristic in this taxon. The remaining species in Table 5 have their own features that separate them from their morphologically close relatives. Valve dimensions are not very useful to differentiate these species and, apart from valve shape, SEM features should be used to distinguish them.

Pseudostaurosira subsalina (Hustedt) E. Morales (in Cejudo-Figueiras et al. 2011, p. 69, figs 2–33, 94–99, 107, 109, 111) is perhaps the closest species to P. occulta sp. nov. at the morphological level (Table 5). However, the former taxon has valves with parallel sides, poorly-developed volae and a single valve mantle areola, larger than the rest of areolae along the same stria. In the case of spines, these have an overall flared shape, somewhat resembling an ice cream cone, spatulate body and tip, the latter bearing two small lateral projections. The apical pore fields in P. subsalina sit on a step-like apex.

Likewise, the remaining species in Table 5 can be readily separated from P. occulta sp. nov. For each taxon we highlight the salient distinguishing features. Pseudostaurosira polonica (Witak & Lange-Bertalot) E. Morales & M.B. Edlund (2003, p. 235, figs 25–32, 45–50) has broadly elliptic valves, sometimes clavate, the areolae are very wide, and the spines are hollow. Pseudostaurosira oliveraiana Grana, E. Morales, Maidana & Ector (in Grana et al. 2018, p. 63, figs 2–15, 16–24) has valves with subcapitate to cuneate ends, trumpet shaped spines, and externally sunken apical pore fields that open interiorly into an elliptical depression but with a raised central area. In turn, P. zolitschkae M.L. García, S. Bustos, Maidana & E. Morales (in García et al. 2021, p. 11, figs 81–91, 103–109) has the widest range of morphological variability of all taxa included in Table 5 producing smaller valves with clearly biconvex sides and pointy ends. The areolae acquire an 8-shape configuration due to the thick origin of the volae that arise from the longer (transapical) axes of the areolae. The spines in this taxon are T-shaped and the apical pore fields are cavernous and small.

Pseudostaurosira linearis has a similar overall shape to P. occulta sp. nov. However, as stated above when comparing it to P. sajamaensis, P. linearis is a fossil species, and it has much wider areolae, has more coarsely striated valves (12–14 striae in 10 m versus 14–16 in P. occulta sp. nov.) and has all the other features cited in the comparison to P. sajamaensis that P. occulta sp. nov. does not possess.

Pseudostaurosira occulta sp. nov. has been reported before under the name “Fragillaria zeilleri Heribaud[sic]” by Servant-Vildary and Roux (1990, p. 276, fig. 29). The shape of the valve imaged under SEM, the characteristics of the areolae and spines resemble what we are describing here as the new species P. occulta. Type material of Fragilaria zeilleri, now Pseudostaurosira zeilleri (Héribaud) D.M Williams & Round (1987, p. 276, no figures), was studied by Serieysol (1988), who showed a taxon with widely elliptic valves and cuneate ends, axial area varying from linear to elliptic, striae composed of narrower areolae, usually one larger after the spine, on the valve mantle. The spines in this taxon have an overall flat, trumpet-like shape without lateral projections or serrate borders. The apical pore fields in P. zeilleri are clearly visible, are somewhat cavernous but lack any external coverings.

Pseudostaurosira oblonga sp. nov. can be distinguished by clearly oblong valve shape, the externally and internally depressed axial area, the trapezium-shaped profile of the spines and the incipient stipules that appear to be facultative (Table 2). This species is clearly different from P. heteropolaris sp. nov. simply based on the small size of the latter and its heteropolar valve shape, although we present other distinguishing features in Table 2.

The remaining species differ from P. oblonga sp. nov in the following salient features, selected from Table 2. Pseudostaurosira alvareziae Cejudo-Figueiras, E. Morales & Ector (in Cejudo-Figueiras et al. (2011), p. 69, figs 34–73, 100–105, 106, 108, 110) characteristically has two larger areolae before and after the spines along the same stria, the stipules are small and conical and the apical pore fields are typically covered by twisted flaps. Pseudostaurosira americana E. Morales (in Cejudo-Figueiras et al. 2011, p. 70, figs 74–93, 112–115. See also Morales et al. 2013b) has a V-shaped middle opening in the spine body, large stipules covering the subtending areolae on the valve mantle, and an apical pore field in which the external openings of the poroids lie in troughs. Pseudostaurosira bardii Beauger, C.E. Wetzel & Ector (in Beauger et al. 2019, p. 4, figs 2–56), on its part, has spines with a triangular profile, spines that have tips with serrate borders.

As it has been seen here, defining Pseudostaurosira as a genus distinguished by short (apical extension) and wide (transapical extension) vimines (relative to the size of the areolae) (Morales et al. 2019b), allows the analysis of the variability of other features for the discrimination of species. That is, there is a large chance that the features of the vimines result in an evolutionary character that defines and separates the genus from others; however, this possibility for vimines to be synapomorphic requires demonstration, and we will perform the necessary cladistic analyses once a fair amount of species have been described and the type material of remaining key species has been studied (e.g. P. pseudoconstruens, P. microstriata (Marciniack) Flower [2005, p. 65], etc.).

As expressed in the descriptions of the new species and the comparative tables presented herein, the salient features that can be used to distinguish species are the features of the axial area, virga and vimines, the areolae and subareolar features (volae, rotae, flaps and internal depositions), the spines (base, body and tips) and stipules, and apical pore fields. We have tried to find differences in other features such as valve shape, morphometric measurements, stria density, blisters and girdle bands, but we have been unsuccessful in finding sufficient variability across a large number of species. As more species are described and type material is re-analyzed, it is possible that the latter characters take more importance in defining species.

This work was co-funded by Portuguese Foundation for Science and Technology (FCT) project UIDB/04683/2020 - ICT (Institute of Earth Sciences), and the Agência Portuguesa do Ambiente, APA-000004DFIN.AALP/2017 integrated within the Operational Program for Sustainability and Efficiency in the Use of Resources 2014-20, POSEUR-03-2013-FC-000001.Funding for this research was also partly provided in the framework of the DIATOMS project (LIST-Luxembourg Institute of Science and Technology). We appreciate the revisions made by Dr. D. M. Williams, and an anonymous reviewer who greatly improved the text.