Testate Amoebae ( Amebozoa : Arcellinida ) in Tropical Lakes of Central Mexico

Testate amoebae are common single-celled eukaryotic organisms in aquatic ecosystems. Despite their important role in these ecosystems, and their potential as bioindicators and paleoindicators, they remain poorly studied in Mexico. The major objectives of this study were to: 1) increase knowledge of testate amoebae in Mexico’s tropical lakes, and 2) create a catalog of high-quality scanning electron micrographs that can be used for future ecological and paleoenvironmental studies. We collected surface-sediment samples from 29 lakes, located in the Transmexican Volcanic Belt, one at each lake during June and October 2011, and March 2013. Sediments were collected with an Ekman grab and preserved in anhydrous ethanol. Sub-samples were observed under a stereomicroscope and morphometric data for each species were recorded. Total diameter and aperture diameter were measured on circular tests. Irregularly shaped tests were measured for length and width of the aperture and for the size of the whole test. If a specimen possessed spines, the length of one randomly selected spine was measured. The best-preserved specimen of each taxon was photographed with an optical microscope and a scanning electron microscope (SEM). We found 41 taxa of testate amoebae belonging to the genera: Arcella, Argynnia, Centropyxis, Cucurbitella, Cyclopyxis, Cyphoderia, Difflugia, Euglypha, Lesquereusia, Pentagonia, Pseudodifflugia and Scutiglypha. Twelve species not previously reported for Mexico were recorded, along with 13 varieties. The average number of taxa recorded in each lake was eight, and the highest taxonomic richness was 18. The taxon found in the greatest number of lakes was Centropyxis aculeata var. aculeata. Taxonomic richness varied among lakes in the same region. This could reflect lake-specific differences in environmental conditions, underscoring the need for more detailed studies that include collection of data on physical and chemical variables in the lakes. Our results highlighted the need of further studies for the distribution patterns and ecology of lacustrine testate amoebae. Rev. Biol. Trop. 64 (1): 393-413. Epub 2016 March 01.

Also referred to as thecamoebians, testate amoebae are single-celled amoeboid protozoa in which the cytoplasm is enclosed within an external shell or discrete test from which pseudopodia emerge (Ogden & Hedley, 1980).These tests range in size from 5 to 300 μm (Smith, Bobrov, & Lara, 2008).Some species have an autogenous test that can be proteinaceous, siliceous or, rarely, calcareous.Other species possess a xenogenous test formed of agglutinated organic or mineral particles captured from the surrounding environment (Ogden & Hedley, 1980;Meisterfeld, 2002aMeisterfeld, , 2002b)).Test morphologies are diverse, which allows identification of these organisms to the species level (Mitchell, Charman, & Warner, 2008).
We studied a database of ~2 800 publications on testate amoebae worldwide (Medioli, Bonnet, Scott, & Medioli, 2003).Results showed that the majority of studies were conducted in temperate, Northern Hemisphere countries.Nevertheless, tropical countries such as Mexico, Brazil, the Democratic Republic of the Congo, Republic of the Congo and India had a few publications on testate amoebae over the last century.The greatest numbers of studies worldwide were of taxonomic nature, and a few in tropical localities were related to ecology or paleoecology.More than the half of the research accomplished in tropical latitudes was conformed by technical publications or unpublished reports.There is thus a knowledge gap regarding thecamoebians in tropical countries, and further studies are needed in these regions.Additionally, most previous taxonomic research in the Neotropics identified species using only light microscopy (Lahr & Lopes, 2006).The increasing use of testate amoebae as bioindicators and paleoindicators has increased the need for further studies to clarify taxonomy, and elucidate distribution patterns and ecology.
The goals of this study were to identify species assemblages of testate amoebae from lakes distributed across central Mexico, in the Northern Neotropics, and produce a catalog of high-quality scanning electron microscope images of those taxa that can be used for future ecological and paleoenvironmental studies.

MATERIALS AND METHODS
Samples were collected from 29 lakes across central Mexico in June and October 2011 and March 2013 (Table 1).The lakes are located within the Neotropical Transmexican Volcanic Belt (Fig. 1).Surface sediment samples were collected with an Ekman grab at the deepest site in each lake (Table 1) and at littoral sites, 0.5 to 1 m deep.In the laboratory, sediment samples were preserved in anhydrous ethanol and sub-samples of 2 cm 3 wet volume were examined in a Petri dish under a stereomicroscope (Zeiss STEMI 2000-C Schott SeriesEasyLED).Testate amoebae tests in each sediment sub-sample were extracted using a fine brush (Ellison & Ogden, 1987).
Total diameter and aperture diameter were measured on circular tests.Irregularly shaped tests were measured for length and width of the aperture and for the size of the whole test.If a specimen possessed spines, the length of one randomly selected spine was measured.Morphometric data were recorded on at least 30 specimens of each species, with preference given to specimens from the same lake.In some cases, however, there were insufficient tests to measure the required 30 specimens from a single lake, in which case we used individuals from different lakes.In the event that the total number of specimens of a taxon, even from multiple sample lakes, failed to yield  The best-preserved specimen of each taxon was photographed.Photographs were taken with an Olympus BX50 optical microscope and Olympus DP11 camera, and a Jeol JSM 5 600-LV LCM scanning electron microscope (SEM).Identification was made using available taxonomic keys for testate amoebae, including Ogden and Hedley (1980), Kumar and Dalby (1998), Lee, Leedale and Bradbury (2000), Charman, Hendon and Woodland (2000) and Escobar, Brenner, Whitmore, Kenney and Curtis (2008).Lacustrine arcellacean species can display a large amount of ecophenotypically controlled morphological variability.It is accepted practice that researchers working in lakes assign informal infrasubspecific 'strain' names for ecophenotypes to avoid possible description of unwarranted new species.Although the International Code of Zoological Nomenclature stipulates that infra-subspecfic-level designations have no status (International Commission on Zoological Nomenclature [ICZN], 1999), they are useful for distinguishing environmentally significant populations in lacustrine environments (Patterson, Roe, & Swindles, 2012).

Species characterization and previous occurrences
Arcella conica Deflandre, 1928 Autogenous brown test.Hexagonal in ventral view with a central, circular aperture.Domed in lateral and dorsal view with depressions forming angular facets bordered by prominent folds and flattened vertices (Fig. 2A).This is the first record of this species in Mexico.

Arcella dentata Ehrenberg, 1830
Autogenous colorless or yellow test.Circular in ventral view, with 9 to 14 spine-like projections that either formed incipiently or were well developed.Aperture: circular and central.Shallow-bowl shaped in lateral view with flattened vertex and 9 to 14 ridges in dorsal view (Fig. 2B, Fig. 2C, Fig. 5A).The species was observed previously in Mexico in a semipermanent freshwater body (Tiscareño, 2008).

Arcella discoides Ehrenberg, 1843
Autogenous test, colorless, yellow, brown, white, red or black.Circular in ventral view with a smooth surface.The aperture is circular, invaginated and bordered by a shallow lip, usually surrounded by a ring of numerous small pores.Plano-convex in lateral view, sometimes arched with a basal border (Fig. 2D, Fig. 2E, Fig. 5B).The species was previously observed in Mexico in lakes, springs and wetlands (López-Ochoterena & Roure-Cane 1970; Bobrov et al., 2013).

Arcella gibbosa Penard, 1890
Autogenous yellow test.Circular in ventral view.Test surface is irregular with numerous small pores between the circular plates.Aperture invaginated, circular and with a distinct lip.Hemispherical in lateral view, with regular depressions on the dorsal surface (Fig. 2F, Fig. 2G, Fig. 5C).The species was observed previously in Mexico in lakes and wetlands (Aladro et al., 2007;Bobrov et al., 2013).

Argynnia triangulata Deflandre, 1936
Xenogenic brown test.Trapezoid shape in lateral view, with the fundus larger than the aperture.Aperture terminal and circular.Test surface smooth with regular and flattened mineral aggregates (Fig. 2K).This is the first record of this species in Mexico.

Centropyxis aculeata
Ehrenberg, 1832 var.discoides Xenogenous yellow or brown test.Ovoid or circular, depressed in ventral view and without spines.Aperture sub-terminal, circular or oval and invaginated.Main body hemispherical in lateral view with pronounced tapering toward the aperture.Test surface is rough and often covered with mineral grains (Fig. 2O, Fig. 2P, Fig. 5H).This variety was observed previously in Mexican cenotes (van Hengstum et al., 2008).

Centropyxis constricta
Ehrenberg, 1843 var.aerophila Xenogenous yellow or brown test.Ovoid in ventral view.Aperture sub-terminal, circular or ovoid, invaginated and with a semi-circular rim.Spherical in lateral view and slightly flattened towards the aperture.The test surface is generally rough with mineral grains or diatom frustules and smooth only in a small region around the aperture (Fig. 2Q, Fig. 2R, Fig. 5I).This variety was observed previously in Mexican cenotes (van Hengstum et al., 2008).

Centropyxis constricta
Ehrenberg, 1843 var.constricta Xenogenous yellow or brown test.Ovoid in ventral view, with 2 or 3 spines on the fundus.The aperture is sub-terminal, ovoid, invaginated and with a semi-circular rim.Spherical in lateral view and slightly flattened towards the aperture.The test surface is rough with mineral grains (Fig. 2S).This variety was observed previously in Mexican cenotes (van Hengstum et al., 2008).

Centropyxis constricta
Ehrenberg, 1843 var.spinosa Xenogenous yellow or brown test.Ovoid in ventral view, with 2-6 spines on the fundus.Aperture sub-terminal, ovoid, invaginated and with a semi-circular rim.Spherical in lateral view and flattened towards the aperture.The test surface is rough with mineral grains or diatom frustules (Fig. 2T, Fig. 2U, Fig. 2V).This is the first record of this variety in Mexico.

Centropyxis deflandrei Rampi, 1950
Xenogenous brown test.Circular in ventral view and laterally depressed.Aperture ovoid to circular and sub-terminal, almost half the size of the test.Without spines.Test surface with mineral grains or diatom frustules (Fig. 2W).This is the first record of this species in Mexico.A similar species (Centropyxis cf.deflandrei), however, was reported previously in soils and wetlands (Bobrov et al., 2013).

Cucurbitella tricuspis Carter, 1856
Xenogenous brown test.Spherical to vaseshaped in lateral view, with a terminal, circular and crenulated aperture.Often with a thin lip surrounded by a 5-11-lobe collar.Test surface with mineral grains or diatom frustules (Fig. 3A, Fig. 3B, Fig. 5J, Fig. 5K, Fig. 5L).This is the first record of this species in Mexico.

Cyphoderia ampulla Ehrenberg, 1840
Autogenous brown test.Ovoid in lateral view, with round base and tapered near the aperture, forming a short, retort-shaped neck.Aperture terminal and circular.The surface test is composed of oval plates on an organic matrix (Fig. 3E, Fig. 5M).Species previously observed in a lake in Mexico (Bobrov et al., 2013).

Difflugia labiosa (Leidy, 1874) Penard, 1902
Xenogenous brown test.Pyriform in lateral view.Fundus tapers slightly to a rounded eccentric apex, with a thin collar surrounding the aperture, which is terminal and circular.Small mineral grains on the surface test (Fig. 3F).This is the first record of this species in Mexico.

Difflugia distenda
Gauthier-Lièvre and Thomas, 1958 Xenogenous test with colorful mineral grains.Almost spherical, with two symmetrical hornlike spines at the fundus.Thin collar with small mineral grains and terminal aperture (Fig. 3G).This is the first record of this species in Mexico.

Difflugia bidens Penard, 1902
Xenogenous brown test.Ovoid and laterally compressed, with 2-5 small spines at the fundus.Aperture terminal, circular and large, without a collar.Some spines can be bifurcated.Test surface with relatively smooth mineral grains (Fig. 3H, Fig. 3I, Fig. 3J).This is the first record of this species in Mexico.

Difflugia fragosa Hempel, 1898
Xenogenous, yellow or brown test.Elongated, with 3-5 large projections running from the middle of the test to the fundus, forming wide spines or tubercles.Aperture terminal and circular, with a thin collar.Test surface formed of small mineral grains (Fig. 3M, Fig. 3N).This is the first record of this species in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.bryophila Xenogenous, brown test.Pyriform in lateral view, with a conical fundus.Aperture terminal and circular, formed by mineral grains cemented together, without a collar.Test made of coarse sand grains (Fig. 3Q, Fig. 3R, Fig. 5P).This is the first record of this variety in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.glans Xenogenous, brown test.Ovoid in lateral view, with a rounded fundus.Aperture terminal, large, circular and formed by mineral grains cemented together, without a collar.Test made of coarse sand grains.This is the first record of this variety in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.lanceolata Xenogenous, brown test.Pyriform in lateral view, with a rounded fundus.Aperture terminal, circular and formed by mineral grains cemented together, without collar.Test made of small mineral grains (Fig. 3S).This is the first record of this variety in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.linearis Xenogenous, brown test.Ovoid and elongated in lateral view, with a conical fundus.Aperture terminal and circular, without a collar, formed by mineral grains cemented together.Test made of small mineral grains (Fig. 3T).This is the first record of this variety in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.oblonga Xenogenous, brown test.Pyriform and elongated in lateral view, with a large neck and rounded fundus.Aperture terminal, circular and formed by mineral grains cemented together.Test composed of angular sand grains and diatom frustules (Fig. 3U, Fig. 3V).This is the first record of this variety in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.spinosa Xenogenous, brown test.Pyriform and elongated in lateral view, with a short or long neck and a spine on the fundus.Aperture terminal, circular and formed by mineral grains cemented together.Test composed of angular sand grains and diatom frustules (Fig. 3W, Fig. 3X).This is the first record of this variety in Mexico.

Difflugia oblonga
Ehrenberg, 1832 var.tenuis Xenogenous, brown test.Pyriform and elongated in lateral view, without a neck and a truncated fundus.Aperture terminal and circular, formed by mineral grains cemented together.Test composed of angular sand grains and diatom frustules (Fig. 4A, Fig. 4B).This is the first record of this variety in Mexico.

Difflugia protaeiformis
Lamarck, 1816 var.acuminata Xenogenous, hyaline to brown test.Cylindrical in lateral view, with slight tapering near the aperture and a dorsal spine sometimes very large and slightly curved.Aperture terminal and circular, with a smooth outline.Test made of mixed-size mineral grains or diatom frustules (Fig. 4C, Fig. 4D, Fig. 4E, Fig. 5Q).This is the first record of this variety in Mexico.

Difflugia protaeiformis
Lamarck, 1816 var.amphoralis Xenogenous, yellow to brown.Elongated test, almost biconical in lateral view, narrowing toward the aperture and with a large projection in the dorsal region that forms one or two wide spines.Aperture terminal and circular, with a smooth outline and a short collar.Test made of coarse grains (Fig. 4F, Fig. 4G).This is the first record of this variety in Mexico.

Difflugia protaeiformis
Lamarck, 1816 var.claviformis Xenogenous, opaque test.Cylindrical in lateral view, slightly tapering near the aperture and with a wide spine in the dorsal region.Aperture terminal and circular, with a smooth outline.Test made of coarse grains (Fig. 4H).This is the first record of this variety in Mexico.

Difflugia urceolata
Carter, 1864 var.elongata Xenogenous, brown and opaque test.Ovoid in lateral view, with a rim sometimes pronounced and curved.Some specimens can have wide spine-like protuberances.Aperture large, terminal and circular, with a relatively smooth outline.Test made of mixed-size grains or diatom frustules (Fig. 4I, Fig. 4J, Fig. 5T).This is the first record of this variety in Mexico.

Difflugia urceolata
Carter, 1864 var.urceolata Xenogenous, brown and opaque test.Spherical in lateral view, with a pronounced rim that curves back on itself.Occasional wide, spine-like protuberances.Aperture large, terminal and circular with a relatively smooth outline.Test made of mixed-sized grains or diatom frustules (Fig. 4K, Fig. 4L).This is the first record of this variety in Mexico.

Euglypha acanthophora
Ehrenberg, 1814 Autogenous hyaline test.Ovoid in lateral view, with a rounded fundus.Aperture terminal and circular.Test composed of oval to hexagonal plates.Those forming the aperture display a serrated, V-shaped edge, with 5-7 teeth bordering each of these margins.Some dorsal-region plates project dorsally like spines, and can be up to three times the size of the plates themselves (Fig. 4M, Fig. 5R).Species previously observed in Mexico in lakes and wetlands (Aladro et al., 2007;Bobrov et al., 2013).

Lesquereusia spiralis Ehrenberg, 1840
Autogenous, brown test.Spherical in lateral view, with an asymmetrical neck and laterally, slightly narrow.Spine-like dorsal projection.Aperture terminal and circular.Test surface composed of numerous curved rods (Fig. 4O, Fig. 5S).This is the first record of this species in Mexico.Gauthier-Lièvre and Thomas, 1958 Xenogenous brown test.Elongated in lateral view, with four short, wide, spine-like projections on fundus.Short collar and terminal aperture.Surface with small mineral grains (Fig. 4P).This is the first record of this species in Mexico.

Pseudodifflugia fulva Archer, 1870
Xenogenous brown test.Spherical in lateral view, with a very irregular shape because of the large agglutinated particles.Aperture circular, large and smooth in ventral view (Fig. 4Q, Fig. 4R, Fig. 4S).This is the first record of this species in Mexico.

Scutiglypha cabrolae Smet and Gibson, 2009
Autogenous test.Plates of different shape depending on the row level.Plates in row around aperture with a triangular edge forming a serrated margin.The opposite end of the plate is semi-circular in shape.Rows of plates increasingly distant from the aperture have decreasingly rounded upper edges, culminating in generally small, but sometimes longer and wider spines in the rows at the fundus (Fig. 4T).This is the first record of this species in Mexico.

Taxa distribution in lakes:
The greatest number of taxa found in a single lake (No. 4) was 18, and only one lake (No. 6) had no individuals.(Species richness in each lake was tabulated in Appendix I).On average, we recorded eight taxa in each lake.The most-observed taxon was Centropyxis aculeata var.aculeata, observed in 22 of the 29 lakes, followed by Arcella discoides, recorded in 17 lakes.In contrast, eight species were recorded in only one lake (Arcella conica, Centropyxis deflandrei, C. ecornis, Cyphoderia ampulla, Difflugia distenda, Lesquereusia spiralis, Pseudodifflugia fulva and Scutiglypha cabrolae).The most similar lakes in terms of species composition were lakes No. 1 and 19, which shared 10 species.Specific richness of testate amoebae in the lakes does not seem to be associated with water depth or lake altitude, so other environmental variables should be explored.only in a recent work (van Hengstum et al., 2008).This may be a consequence of the fact that researchers in several previous studies in Mexico identified individuals only to species level, whereas in recent studies, identifications were made to the variety level.
Lakes of the same region displayed different species richness.This could indicate differences in environmental conditions, underscoring the need for detailed studies that include collection of data on physical and chemical variables in the lakes.
In a brief review of testate rhizopod studies in lakes around the world, we noted that the species assemblages we report from Central Mexico resemble most closely assemblages from Neartic water bodies close to Lake Ontario, and near other Canadian lakes (Patterson, MacKinnon, Scott, & Medioli, 1985;Patterson, Barker, & Burbidge, 1996;Reinhardt, Dalby, Kumar, & Patterson, 1998;Kumar & Patterson, 2000;Torigai, Schröder-Adams, & Burbidge, 2000;Roe, Patterson, & Swindles, 2009;Roe & Patterson 2014).In those studies, the composition and abundance of species generally indicated mesotrophic conditions and pollution, suggesting it will be important to gather such information on Mexican lakes to see if testate amoebae can serve as bioindicators of these variables in those water bodies.
The high number of new records provided by this study shows the scarcity of information regarding the diversity of testate amoebae in lakes of Mexico.The morphometric data suggest that new varieties could be described in lakes throughout the center of the country.Only through a comprehensive species characterization will it be possible to carry out ecological and palaeoecological studies in the future.

Fig. 1 .
Fig. 1.Location of the 29 study lakes in Mexico.
Ehrenberg, 1832 var.aculeata Xenogenous yellow or brown test.Ovoid or circular and depressed in ventral view, with 4-10 posterolateral spines.Aperture subterminal, circular or oval and invaginated.Capshaped in lateral view and tapers toward the aperture.Test surface is smooth only around the aperture and is covered near the spines and on the dorsal surface with mineral grains or diatom frustules (Fig. 2L, Fig. 2M, Fig. 2N, Fig. 5F, Fig 5G).This variety was observed previously in Mexican cenotes (van Hengstum et al., 2008).