To extract sponge spicules from sediments, the wet oxidation methodology is often used (e.g., Battarbee, 1986; Harrison, 1988; Volkmer-Ribeiro and Turcq, 1996; Figure 1). Through this method, it is possible to extract and prepare other siliceous microfossils (e.g., plant phytoliths and diatoms) as well. The extraction steps are as follows:

This guide has been designed based on the available taxonomic literature for the Neotropical region, including the World Porifera Database (De Voogd et al., 2022), personal collections of the authors, and microscope slides archived in the Porifera Laboratory (LABPOR) at Pernambuco Federal University (Recife, Brazil). This guide does not replace the need to consult the proper taxonomic keys, as well as the literature describing the species since morphological variations are possible and cannot be fully explored here. We describe here the spicules of 27 genera of freshwater sponges from the Neotropical region (Figures 3–14). Freshwater sponges (Neotropics fauna) were identified based on their morphology, exception given to the genus Rosulaspongilla (Sokolova et al., 2021), which was identified based on DNA (Supplementary Material 1). Some genera may have morphological differences or similarities when compared to species from other zoogeographic regions. In total, 15% of the species found in the Neotropical region also occur in other areas of the world, such as in the Oriental Region (OL), Nearctic Region (NA), Palaearctic Region (PA), Australian Region (AU), and Afrotropical Region (AT) (Supplementary Material 1).

The skeleton of freshwater sponges consists of a fibrous network of spongin and siliceous spicules (also known as the spicular complement). The megascleres are relatively large and form the main structural component of sponges. In contrast, microscleres are relatively small and often lack a structural function when present (Manconi and Pronzato, 2002). Megascleres can be oxeas (monaxon diactinal spicule pointed at both ends) to strongyles (an isodiametric, diactinal megasclere with rounded end), smooth, spiny, or granular, occasionally with larger tuberculate ornamentations, with sharply pointed, rounded, or occasionally inflated points liked tylote (diactinal megasclere with swelling on each end) (Boury-Esnault and Rützler, 1997; Manconi and Pronzato, 2002; Figure 2). Microscleres can be present or absent, including smooth or spined oxeas or strongyles, aster-like (spicules in which the rays radiate from a central point), and pseudobirotules (birotules that have curved hooks at the ends) spicules. Freshwater sponges can also produce gemmules, a resistant body and asexual propagule produced by Spongillidae, Metaniidae, and Potamolepidae, to survive potentially stressful seasonal environmental conditions (e.g., desiccation and ice-cover). This body is armed with gemmuloscleres and is the most important structure for species identification (Manconi and Pronzato, 2002). Gemmuloscleres encompass various morphologies, ranging from oxeas to strongyles, birotules (a type of spicule with a straight shaft and umbrella-shaped ends), pseudobirotules (does not show a developed rotule but a group of curved hooks radiating from the apices of the shaft), sanidasters (straight spicule having spines at intervals along the entire length). Spines along the shaft are perpendicular to the axis and may or may not be spirally arranged (those at the ends diverge obliquely), parmuliform (spicule with a single rotule supporting a short acute conical stem) to boletiform-tubelliform (characterized by a proximal large, irregularly circular, flat rotule with the entire margin supporting a smooth shaft decreasing in thickness toward the distal apex), and shaped as umbonate (pseudo-rotule with large hooks bearing microspines at their apices) forms (Boury-Esnault and Rützler, 1997; Manconi and Pronzato, 2002).

It is important to differentiate the categories of spicules present in the sediment if they are megascleres, microscleres, and/or gemmuloscleres. It is preferable for the analyst to measure the size range of the spicule morphotype (e.g., length, width, and diameter) and include illustrations or scanning electron microscope (SEM) images to generate the most robust characterizations.

Important remarks regarding some sponge species that should be considered:

The species Arinosaster patriciae Volkmer-Ribeiro, Tavares-Frigo, Ribeiro & Bichuette, 2021, presents megascleres long, robust, smooth, and abruptly pointed oxea. Gemmuloscleres have not been detected. Microscleres fall into three categories; the most abundant are the heavily spined oxea; spines can be straight or curved, and simple or compound (have a bouquet-like structure); rare microscleres include euasters, spherasters, and spheres in more than one size category, as well as rare smooth to spined spirasters. These microscleres were detected only in the preparations containing the dissociated spicules (Volkmer-Ribeiro et al., 2021). The highlighted spicules have a strong similarity to phytoliths, namely, SPHEROID ECHINATE, SPHEROID ORNATE, and SPHEROID PSILATE (Neumann et al., 2019). Phytoliths are structures of silica accumulated by plants and are also preserved in sediments. Their postmortem and their morphologies are directly related to specific botanical families (Piperno, 2006). The SPHEROID ECHINATE morphotype is most often associated with palms (Arecaceae) and Bromeliaceae (Albert et al., 2009; Strömberg et al., 2013); the SPHEROID ORNATE are used as indicators of woody vegetation, and SPHEROID PSILATE have been used as evidence of non-grass plants (Strömberg, 2004, 2005; Strömberg et al., 2018). Concerning Arinosaster patriciae, images of this genus are not included in this guide, as confusion remains regarding the identification of the spicules and their differentiation from phytoliths.

The Neotropical zoogeographic region has the highest species richness. Here, we provide a taxonomic description and information for the genera of freshwater sponges in the Spongillidae, Metaniidae, and Potamolepidae families, and the incertae sedis genus. For each genus, we present a list of species, drawings of gemmuloscleres and/or microscleres that attribute specific taxonomic characteristics, and the appropriate bibliography for reference.

Spongillidae Gray, 1867.

Spicules: Megascleres fall into one category (acanthoxeas or smooth oxeas) or two categories (alpha megascleres, acanthoxeas or smooth oxeas; and beta megascleres, acanthoxeas, and acanthostrongyles). Microscleres are absent. Gemmuloscleres are acanthoxeas, acanthostrongyles, pseudobirotules with a spiny shaft, and bent smooth hooks, acanthoxeas, and grading from long to very short, from stout to slim, acanthostrongyles and acanthoxeas (Figures 3A–E).

Species information: Bonetto and Ezcurra de Drago (1970), Manconi and Pronzato (2005), Buso et al. (2012), Calheira and Pinheiro (2018).

Spicules: Megascleres are oxeas, generally smooth, sometimes irregularly microspined. Microscleres are pseudobirotules with long hooks, pseudobirotule with short hooks, and/or acanthoxeas varying in shape and size, with a variable number of spines, simple (straight or curved) and/or compound (Figures 3F–L). Gemmuloscleres are birotules with rotules microspines on their margins, convex to flat or sanidasters (Figures 3M–S).

Species information: Bonetto and Ezcurra de Drago (1966), Ezcurra de Drago (1974b), Pinheiro et al. (2015b), Calheira and Pinheiro (2016).

Spicules: Megascleres are predominantly microspined strongyles, and rarely microspined strongyles. Microscleres are pseudobirotules with four to seven radial hooks on each pseudorotule and a smooth shaft (Figure 3Q). Gemmuloscleres are microspined strongyles and acanthoxeas (Figures 3R, S).

Species information: Bonetto and Ezcurra de Drago (1966), De Rosa-Barbosa (1988), Manconi and Pronzato (2002), Pinheiro et al. (2013).

Spicules: Megascleres are smooth oxeas or with microspines. Microscleres range from aster from simple acerates with one or more long divergent branch spines to true “euasters” with spiny rays (Figures 4A–C). Gemmuloscleres are birotules, with straight cylindrical shafts and large spines (Figures 4D, E). Rotules vary from flat to slightly umbonate with margins bearing numerous small blunt and recurved teeth or spines.

Species information: Potts (1885), Penney and Racek (1968), Volkmer-Ribeiro (1992), Manconi and Pronzato (2002), Cândido et al. (2010).

Spicules: Megascleres are oxeas that range from smooth to microspined. Microscleres are absent. Gemmuloscleres are birotules with smooth or spined shafts (simple and compound spines), with secondary spines (Figures 4F–L). Flat rotules with incised irregular margins may be present.

Species information: Penney and Racek (1968), Ezcurra de Drago (1975a), De Rosa-Barbosa (1979), Ricciardi and Reiswig (1993), Manconi and Pronzato (2002), Pisera and Sáez (2003), Pinheiro et al. (2004), Nicacio and Pinheiro (2015).

Spicules: Megascleres are smooth or spined oxeas. Microscleres absent. Gemmuloscleres are smooth oxeas or acanthoxeas to acanthostrongyles (Figures 5A–C).

Species information: Penney and Racek (1968), Ezcurra de Drago (1974a), Manconi and Pronzato (2002), Nicacio and Pinheiro (2015).

Spicules: Megascleres are acanthoxeas. Microscleres are acanthoxeas, with spines that can be straight or curved, and simple or compound with a bouquet-like structure (Figures 5D–F). Gemmuloscleres fall into one or two categories: pseudobirotules (with teeth projecting from the center of the pseudorotule and can be simple or anastomosing) (Figure 5G), and birotules, and shafts with conical spines (simple or compound) (Figures 5H, I). Rotules are smooth or covered in microspines, circular, convex, and identical, with microspines on their margins, and the margins can be serrated. Heteromeyenia horsti is a possible synonym of H. insignis (Pinheiro et al., 2015a; Calheira et al., 2020).

Species information: Manconi and Pronzato (2002), Batista et al. (2007), Pinheiro et al. (2015a).

Spicules: Megascleres oxeas are microspined except at the ends. Microscleres are absent. Gemmuloscleres birotules fall into two categories (long and short), with rotules flat and microspined, one always larger than the other (Figures 6A–F). Birotules have long margins with serrations, and birotules exhibit short margins that range from serrated to toothed.

Species information: Volkmer-Ribeiro and Motta (1995), Manconi and Pronzato (2002).

Spicules: Megascleres are smooth to spined oxeas. Microscleres are acanthoxeas, microspine-bearing along their length, with conical projections that are also microspined (Figures 6G–K). Gemmuloscleres are absent. Gemmules are missing pneumatic layers and gemmuloscleres, with the megascleres forming cages to contain the gemmules, or else they irregularly adhere to the gemmular wall.

Species information: Volkmer-Ribeiro and Maciel (1983), Manconi and Pronzato (2002), Volkmer-Ribeiro et al. (2010c).

Spicules: Megascleres are acanthoxeas. Microscleres are absent. Gemmuloscleres are present in two types: birotules (short thin or robust shafts with smooth or with few spines) and pseudobirotules (long and spiny shafts) (Figure 7). Birotules can have rotules with small slightly umbonate to flat and deeply cut into several microspined long rays.

Species information: Volkmer-Ribeiro et al. (1988), Manconi and Pronzato (2002), Volkmer-Ribeiro et al. (2010a), Hernández and Barreat (2017), Gómez et al. (2019).

Spicules: Megascleres are spined oxeas, rarely spined strongyles. Microscleres are absent. Gemmuloscleres are acanthostrongyles with tips bearing apical spines, where spines are concentrated at the tips; they are curved, forming hooks directed toward the center of the spicule, or are straight and sharp, which can form small umbonate rotules (pseudorotules). Pseudobirotules with spiny shafts and bent long spines are at the apices (Figures 8A–C).

Species information: Penney and Racek (1968), Ezcurra de Drago (1975b), Volkmer-Ribeiro and Maciel (1983), Ricciardi and Reiswig (1993), Bass and Volkmer-Ribeiro (1998), Manconi and Pronzato (2002), Volkmer-Ribeiro and Machado (2009), Nicacio et al. (2011).

Spicules: Megascleres are predominantly smooth oxeas. Microscleres are fusiform acanthoxeas densely spined with complex spines in the middle and simple spines at the tips (Figure 8E). Gemmuloscleres are acanthoxeas with large, curved spines more thickly accumulated at the tips and often form mace-shaped structures (Figure 8D).

Species information: Penney and Racek (1968), Manconi and Pronzato (2005), Volkmer-Ribeiro and Machado (2009), Pinheiro et al. (2015c), Sokolova et al. (2021).

Remark: Sokolova et al. (2021) demonstrated from genetic analysis and confirmed with morphological data that the part of the genus Spongilla, “S. alba group” should be separated into another genus, Rosulaspongilla, which reveals a new group of brackish-water sponges. Thus, Spongilla alba from the Neotropical region now is accepted as Rosulaspongilla alba (junior synonym).

Spicules: Megascleres are smooth oxeas. Microscleres are absent. Gemmuloscleres are acanthoxeas with conical spines (Figure 8H) and short birotule with smooth shafts (Figures 8F, G). Rotule with smooth margins of the same size (predominant) or different size (rare) perforated on both sides by the shaft end.

Species information: Volkmer-Ribeiro (1976), Manconi and Pronzato (2002).

Spicules: Megascleres are stout, smooth oxeas, with abruptly pointed extremities. Microscleres are slender, slightly curved, spiny oxeas, all spines capped by a rosette of microspines (Figure 8J). Gemmuloscleres are short, stout, spiny oxeas, straight to slightly curved, covered by large spines curved toward the middle part of the spicule, and their extremities split into a few smaller spines (Figure 8I).

Species information: Penney and Racek (1968), Manconi and Pronzato (2002), Volkmer-Ribeiro and Machado (2009).

Spicules: Megascleres are spined or smooth strongyles and/or oxeas. Microscleres are absent. Gemmuloscleres are birotules with a short stout smooth shaft (Figure 9). The rotules can be with equal or unequal diameters, or only with only one rotule (the other rotule is vestigial) and are circular with entire margins.

Species information: Bonetto and Ezcurra de Drago (1964), Bonetto and Ezcurra de Drago (1965), Penney and Racek (1968), Bonetto and Ezcurra de Drago (1973b), Volkmer-Ribeiro (1973), Volkmer-Ribeiro and De Rosa-Barbosa (1985), Batista et al. (2007), Manconi and Pronzato (2002), Nicacio and Pinheiro (2015).

Metaniidae Volkmer-Ribeiro, 1986.

Spicules: Megascleres are smooth or microspined strongyles to smooth oxeas. Microscleres are absent. Gemmuloscleres present as two types: tubelliform (Figure 10A) and pseudobirotules (Figures 10B, C). Tubelliforms have a proximal large irregularly circular flat rotule with an entire margin supporting a smooth shaft, decreasing in thickness toward the distal end and shaped as an umbonate knob-like rotule with few teeth. Pseudobirotules have cylindrical shafts and hooks of umbonate pseudo-rotules stout and notably recurved bearing microspines at their apices.

Species information: Volkmer-Ribeiro and De Rosa-Barbosa (1972), Manconi and Pronzato (2002).

Spicules: Megascleres are oxeas, generally smooth. Microscleres fall into one or two categories: in the first case with microbirotules straight, with smooth or spiny axis, with regularly cropped rotules, and in the second case with strongly curved and reduced pseudobirotule-like rotules (Figures 10D–I). Gemmuloscleres with one or two categories of pseudobirotules (Figures 10J–M). Pseudobirotules with long, smooth, delicate shafts, and strongly umbonate rotules, are dissimilar in size and can be cut out in a variable number of hooks or teeth, and these are with irregular dispositions. Pseudobirotules are small thick, conspicuously umbonate, usually well-formed, and bearing at their edge six small or quite large, incurved hooks. However, this rotule may be reduced to a knob with a few irregularly formed hooks or spines.

Species information: Manconi and Pronzato (2002), Volkmer-Ribeiro et al. (2005).

Spicules: Megascleres fall in two size classes: alpha megascleres range from smooth straight to curved oxea with abruptly pointed extremities to nanospined and conspicuously curved strongyles, and beta megascleres, when present, usually are curved oxea, two-thirds of the size of the alpha megascleres, and may bear sparse spines. Microscleres are minute, straight to curved, slender to thick spiny oxea with harpoon-shaped extremities and the central portion bearing few to several larger spines; or they are straight, uniformly nanospined oxea (Figures 11A–E). Gemmuloscleres are minute, flat, or umbonate parmuliform spicules with circular or ellipsoid outlines; both faces have a smooth or outer face with a central conical or rounded projection, or a shallow crest and thin or thick, incurved borders (Figures 11F–J).

Species information: Bowerbank (1863), Weltner (1895), Bonetto and Ezcurra de Drago (1968a), Bonetto and Ezcurra de Drago (1973a), Volkmer-Ribeiro and Mothes de Moraes (1981), Volkmer-Ribeiro and Tavares (1995), Manconi and Pronzato (2002), Volkmer-Ribeiro et al. (2017).

Spicules: Megascleres range from oxeas to strongyles that range from densely spined to smooth, particularly at the apices. Microscleres range from acanthostrongyles to oxeas and acanthoxeas, with long perpendicular or slanting spines to aster-like shaped spicules (Figures 12A–E). Gemmuloscleres are stout microspined birotulates (Figures 12F–H).

Species information: Bonetto and Ezcurra de Drago (1966), Manconi and Pronzato (2002).

Remark: Bonetto and Ezcurra de Drago (1966) erected Houssayella and suggested this genus is closely related to the Dosilia Gray, 1967, since both present aster-like microscleres. Volkmer-Ribeiro and Rützler (1997) erected Pachyrotula and suggested Houssayella and Heterorotula closely related to a new genus, due to the similar spicular set.

Spicules: Megascleres fall into two distinct classes: alpha megascleres range from smooth, stout oxea to strongyles, and beta megascleres, when present, range from spiny oxea to strongyles. Microscleres fall into two distinct classes: spiny minute oxea displaying large spines in the middle, with simple and compound spines, sometimes grouped in rosettes, and can show a microgranulation at the extremities (Figures 12I–K). The large spines have lanceolate endings. Gemmuloscleres are boletiform with shafts long to short, smooth to spined, with a variable number of spines, and a collar of spines under the lower rotule (Figures 12L–N). Lower rotules are large, stout, and polygonal, with curved, undulated margins. Upper rotules are knob-like, smooth, or with a few recurved, irregularly placed spines or hooks, or approaching a true rotule with marginal incurved spines.

Species information: Bowerbank (1863), Carter (1881), Volkmer-Ribeiro (1979), Volkmer-Ribeiro (1984), Volkmer-Ribeiro and Costa (1992), Manconi and Pronzato (2002), Castello-Branco et al. (2015).

Potamolepidae Brien, 1967.

Spicules: Megascleres fall into two categories: alpha megascleres are acanthotylostrongyles, with spines grouped in small spots or forming half rings on the convex section of the spicule, the tylote extremities entirely covered with minute spines, or they can be strongyles microgranulated with inflated tips (Figure 13A). Beta megascleres are acanthostrongyles, with microspined tubercules along the spicule length except at the extremities, which are invariably covered with minute spines (Figure 13B). Microscleres are unknown or absent. Gemmules are unknown.

Species information: Volkmer-Ribeiro et al. (2009), Pinheiro et al. (2020).

Spicules: Megascleres can be microspined, smooth or spines oxeas, and/or strongyles microgranules, smooth or covered by spines (Figures 13C–J). Microscleres are absent. Gemmuloscleres are highly variable from smooth oxea curved in the shape of a boomerang to microspined oxeas with variable shapes (curved, with or without pointed tips, folded, completely microspined irregularly spherical). They could be smooth strongyles that may present with an inflated medial portion to spined strongyles, with spines concentrated mainly on the extremities. Irregular strongyles also can be present; spiny strongyles resembling peanut shells. Spines are more abundant at the tips. A bumped middle region is present in the gemmuloscleres so that quite cylindrical ones are rare. Spherical forms are rare.

Species information: Carter (1881), Volkmer-Ribeiro (1963), Bonetto and Ezcurra de Drago (1967), Bonetto and Ezcurra de Drago (1968b), Bonetto and Ezcurra de Drago (1970), Volkmer-Ribeiro (1970), Bonetto and Ezcurra de Drago (1973a), Tavares and Volkmer-Ribeiro (1997), Manconi and Pronzato (2002), Tavares-Frigo et al. (2015).

Potamophloios Brien, 1970 [1969].

Spicules: The neotropical species has megascleres that are large, stout, smooth, straight to curved or angular strongyles, which initiate as slim sharply pointed oxea. Microscleres are absent. Gemmuloscleres vary from straight to curved, smooth, small, stout strongyles, with smaller examples reaching a spheric shape (Figures 13K–M).

Species information: Manconi and Pronzato (2002), Volkmer-Ribeiro et al. (2010b).

Spicules: Megascleres are stout and slightly curved, from smooth to uniformly granulated strongyles with inflated tips. Rare oxeas with blunt tips are also present. Microscleres are slender, slightly curved acanthoxeas (reported as tornotes in the original description) entirely ornamented by tubercules or spines with microspines arranged in rosettes (Figure 13N). Gemmuloscleres are spherical to sub-spherical, most often ellipsoid sterrasters, with an irregular surface apart from a unilateral smooth area (Figure 13O).

Species information: Volkmer-Ribeiro and De Rosa-Barbosa (1978), Manconi and Pronzato (2002), Volkmer-Ribeiro and Parolin (2005).

Spicules: Megascleres range from stout curved strongyles with granulated surfaces and a very thin axial canal, to stout smooth oxeas. Microscleres are absent. Gemmuloscleres are smooth curved strongyles, spherules of silica, and deformed strongyles are also present (Figures 13P, Q).

Species information: Bonetto and Ezcurra de Drago (1964), Bonetto and Ezcurra De Drago (1969), Manconi and Pronzato (2002), Nicacio and Pinheiro (2015).

Spongillidae incertae sedis (Manconi & Pronzato, 2002).

Spicules: Megascleres are oxeas ranging from slender to stout, from straight to slightly curved, from smooth to spined, with acerate tips. Microscleres and gemmules are absent (Figures 14A, B).

Species information: Boury-Esnault and Volkmer-Ribeiro (1991), Manconi and Pronzato (2002).