Ultrastructure of the rotifer integument: peculiarities of Sinantherina socialis(Monogononta: Gnesiotrocha)

Rick Hochberg,1,a Adele Hochberg,1 and Courtney Chan2

1 Department of Biological Sciences, University of Massachusetts Lowell, Lowell, Massachusetts 01854, USA2 Andover High School, Andover, Massachusetts 01810, USA

Abstract. The rotifer integument is a well-described syncytium that contains an apical in-tracytoplasmic lamina (ICL) that functions for both skeletal support and muscle insertion.To date, there is limited information on the structure of the integument in species of Gnesio-trocha, a diverse subclade of Monogononta that consists of solitary, colonial, sessile, andplanktonic species. In this study, we examined the ultrastructure of the integument in thecolonial rotifer Sinantherina socialis to determine how it corresponds to that of othermonogononts. The integument of S. socialis was broadly similar to that of other rotifers,consisting of a thickened glycocalyx, multilaminate ICL, and syncytial epidermis. However,it was different in several regards. The ICL consisted of three distinct layers from apical tobasal: layer 1 consisted of at least two electron-dense laminae; layer 2 was a thickenedmatrix of amorphous, electron-dense material or was fibrous; and layer 3 was an electron-dense lamina of varying thickness that covered the underlying syncytium. Significantly,layers 1 and 2 formed a ridge-and-groove like system of finger-like projections across thetrunk surface that has not been observed in other rotifers. A voluminous syncytial cyto-plasm (up to 3 lm thick) was present beneath the ICL and was mostly electron lucent andwith few organelles. Bundles of potential microtubules were scattered throughout the syncy-tium. We hypothesize that the voluminous cytoplasm with microtubules serves as skeletalsupport for the rotifer’s sessile lifestyle, while the external ridges may function as a texture-based deterrent to predators, or serves to trap secretions from the species’ defensive glands.Basally, the epidermis was highly folded and bordered by a thin basal lamina that separatedthe plasmalemma from the blastocoel. Membrane-bound vesicles were present throughoutthe integument’s cytoplasm and are hypothesized to function in the secretion of extracellu-lar matrix and in the maintenance of the ICL.

Additional key words: cuticle, defense, epidermis, Rotifera, ultrastructure

Rotifera consists of mostly microscopic, free-living invertebrates that inhabit the plankton andbenthos of freshwater lakes, where they form animportant part of the microbial loop (Wallace et al.2015). Two defining rotiferan features, a complexciliated head (corona) and a muscular pharynx withintricate jaws (mastax), have been examined indetail across the three major clades, Bdelloidea,Monogononta and Seisonacea, and have beenimportant in defining their systematics and inter-preting their evolutionary history. A third feature,and one that links the Rotifera sensu strictu withparasitic Acanthocephala (Rotifera sensu lato), is

the structure of the integument (Lorenzen 1985),which is generally described as a syncytium with aninternal “cuticle” of fibrous proteins that create anintracytoplasmic lamina (ICL). Significant variationis present in the ultrastructural organization of theICL across all four clades including the Bdelloidea(Koehler 1965, 1966; Storch & Welsch 1969;Cl�ement & Wurdak 1991), Monogononta (Koehler1966; Cl�ement 1969, 1977, 1980; Storch & Welsch1969; Brodie 1970; Cl�ement & Wurdak 1991),Seisonacea (Ahlrichs 1997), and Acanthocephala(reviewed in Dunagan & Miller 1990). While thefunction(s) of this variation is largely speculative,the integumental organization is still consideredhomologous throughout Rotifera (Cl�ement &Wurdak 1991; Funch et al. 2005).

aAuthor for correspondence.E-mail: rick_hochberg@uml.edu

Invertebrate Biology x(x): 1–8.© 2015, The American Microscopical Society, Inc.DOI: 10.1111/ivb.12085

Cl�ement & Wurdak (1991) have provided themost comprehensive review of integumental ultra-structure throughout the Rotifera (excludingAcanthocephala), and described the epidermis as aform of thickened peripheral skeleton covered bya thin extracellular cuticle (glycocalyx) that itselfserves no skeletal function. The epidermis isentirely syncytial and generally contains a pair ofdistinct laminae. In bdelloids, the laminae includea thin outer electron-lucent layer and a thickerinner electron-dense region, the latter of whichfunctions in skeletal support. In monogononts, thetwo laminae are reversed in organization and aremore diverse in structure, with a variable butthickened electron-dense outer region and a thin-ner and more electron-lucent inner region. Insesionoids, the more basal of the two laminae isthickest (as in bdelloids) and separated from theapical lamina by a thin region of cytoplasm.Membrane-bound vesicles are present throughoutthe integumental cytoplasm of most species andexpel secretion products to the outside of thebody, although the nature of the secretions andtheir functions are unknown.

To date, most studies of the rotifer integumenthave focused on the solitary free-living forms, andthere are no formal descriptions of the integumentin sessile or colonial species that make up the bulkof the diversity within the subclade Gnesiotrocha(Monogononta). While the monophyly of Gnesio-trocha remains speculative, the lineage contains adiversity of species with peculiar lifestyles andbody plans (reviewed in Wallace et al. 2015), thelatter of which may present unusual features suchas the absence of a corona (adults of Collotheca-ceae), the presence of locomotory appendages(e.g., some species of Flosculariaceae), the pres-ence of a hard or soft extracorporeal tube (e.g.,species of Flosculariacea and Collothecacae), orthe presence of defensive epidermal spines orwarts (e.g., species of Sinantherina). It is this latterpeculiarity—unusual surface features—that is thefocus of the current investigation. Hochberg &Lilley (2010) provided a description of the neuro-muscular system of Sinantherina socialis (LINNAEUS1858), a sessile rotifer that forms large colonies onsubmerged vegetation. In their study, the authorsrevealed that the integument of S. socialis had anunusual surface microtexture not documented forany other rotifer. In this study, we provide a moredetailed description of this unusual integumentusing electron microscopy, and speculate on itspotential functions.

Methods

Sinantherina socialis was collected on submergedplants in Mascuppic Pond in Tyngsboro, MA in thesummer months of 2013 and 2014. Living specimenswere photographed with a Sony Handycammounted on either a Zeiss Stemi stereomicroscopeor Zeiss A1 compound microscope. Individual speci-mens were extracted from colonies with “000” insectpins and then anaesthetized with 1.5% MgCl2 inpond water for 10–15 min in a 5 mL embryo dish.

For scanning electron microscopy (SEM), ten col-onies still attached to their substratum (grass) wereanesthetized, fixed in 2.5% glutaraldehyde in0.1 mol L�1 phosphate buffer (PB, pH 7.3) for 2 h,rinsed in 0.1 mol L�1 PB for 1 h, postfixed in 1%OsO4 in 0.1 mol L

�1 PB for 1 h, and rinsed in0.1 mol L�1 PB for 1 h. Colonies were dehydratedin an ethanol series (70%, 90%, 100% 92) for10 min each step, and subsequently critical pointdried in a Tousimis SamDri PVT-3D, coated withgold, and viewed on a JEOL JSM 6390 SEM.

Approximately fifty individual specimens fortransmission electron microscopy (TEM) were fixedin 2.5% glutaraldeyde in 0.1M sodium cacodylatebuffer (pH 7.3) for 2 h, postfixed in 1% OsO4 in0.1 M sodium cacodylate for 1 h, and subsequentlyrinsed in the same buffer for 1 h. Following an eth-anol dehydration (70%, 90%, 100% 92), specimenswere placed in propylene oxide (PO) for 20 min(92), and slowly infiltrated in a PO:epoxy resin mix-ture (Araldite, EMbed 812; Electron MicroscopySciences) in ratios of 3:1 for 1 h, 1:1 for 1 h, and1:3 for 1 h. Specimens were placed in pure epoxyresin overnight (12 h) on a rotator and then embed-ded in pure epoxy resin the next day. The resin wascured in an oven at 60°C for 24 h, and five speci-mens were sectioned at 70–90 nm on a Reichertultracut ultramicrotome. Sections were stained inuranyl acetate and lead citrate and examined on aPhillips EM 400T electron microscope equippedwith an Advantage HRL-B bottom mounted 1.3Megapixel CCD camera and a Pentium computer.All image capture was digital, and no alterations tothe photographs were made except for cropping andchanges to brightness and contrast.

Results

External morphology

Colony members had a smooth body wall whenexamined with brightfield microscopy (Fig. 1A,B).

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Traces of the ridge-like microtexture were barely visi-ble with low magnification SEM (Fig. 1C) but couldbe readily observed at higher magnifications on thedorsal side (Fig. 1D) and ventral side (Fig. 1E,F). Pos-teriorly, the integument of the foot (between the site ofsubstratum attachment and the oviferon; see Fig. 1C)was wrinkled and without noticeable texture (data notshown). The integument of the trunk had a very dense

ridge-and-groove like microtexture that formed longi-tudinal lines along the surface but with numerousinterdigitations between lines that gave them a zipper-like appearance at high magnification (Fig. 2A). Theonly trunk regions that lacked the ridge-and-groovemicrotexture were the openings to the ventral anten-nae, which were formed of a ring of smooth integu-ment around the sensory cilia (arrow, Fig. 1F).

Fig. 1. Adults of Sinantherina socialis. A. Brightfield image of colony on submerged vegetation. Defensive warts (*)are present on all specimens. Scale bar=500 lm. B. Brightfield image of a single adult with eggs (e.g) extracted fromthe colony and showing a smooth body wall. The corona (cr) and trunk (tr) are visible, while the foot is contractedand partly hidden by the eggs. Scale bar=100 lm. C. Scanning electron micrograph (SEM) of a single adult within acolony. The texture of the integument appears less smooth with increasing magnification. The ridged microtexture isonly apparent on the trunk (tr) and around the oviferon (ov), but is absent from the foot (ft) and apical field (af) ofthe corona. Scale bar=500 lm. D. SEM of the anterior trunk between the oviferon and coronal cilia (cc), dorsal view.Scale bar=20 lm. E. SEM of a ventral antennae (va) with cilia (ac) extending out the apical pore. Scale bar=4 lm.F. SEM of the ventral integument posterior of the corona (not shown) and buccal field (bf). The only regions of theintegument that lack microtexture are the openings to the ventral antennae (arrow). Scale bar=10 lm.

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Anteriorly, the surface below the coronal cilia and oneither side of the buccal field also had a ridged integu-ment (Fig. 1F). The apical field of the corona wassmooth and lacked microtexture (Fig. 1C).

Ultrastructure of the integument

Intracytoplasmic lamina. The ridge-and-groove-likemicrotexture of the integument (Fig. 2A) was a prod-

Fig. 2. Ultrastructure of the integument of an adult specimen of Sinantherina socialis. A. Scanning electron micrographof the trunk epidermis showing the ridge-and-groove microtexture. Scale bar=7 lm. Inset: closeup of the integumentshowing the zipper-like appearance. Inset scale bar=3 lm. B. Transmission electron micrograph (TEM) of an obliquesection through the anterior trunk region with the corona retracted into the body. The cilia (ci) and epidermal ciliarycushions (ce) can be seen inside the blastocoel (bc). The ridge-and-groove microtexture of the intracytoplasmic lamina(ICL) is present around this portion of the trunk. Scale bar=8 lm. C. TEM of the ICL showing the three layers: layer1(L1) is the apical cell border; layer 2 (L2) is the mostly granular matrix; and layer 3 (L3) is the electron-dense layerthat borders the underlying cytoplasmic syncytium (sy). A vesicular bulb (v) is proximal to L2, but its function (exocy-totic or endocytotic) is unknown. A thickened basal lamina (bl) is present below the basal plasmalemma (bp). Scalebar=700 nm. D. TEM of the folded body wall of the anterior trunk region. The folds show the stretched regions of theICL, which highlight the fibrous matrix of L2 in this region. L3 is present as a very electron-dense membrane. Scalebar=1 lm.

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Fig. 3. Transmission electron micrographs (TEMs) of sections through the integument of Sinantherina socialis.A. Close-up of the ridge-and-groove system showing the three layers of the ICL (L1, L2, L3). Electron-dense dots(arrowheads) are present close to the base of L1 or within the matrix of L2. The glycocalyx is present as a thickenedextracellular cuticle (cu). Scale bar=150 nm. B. TEM of a section through the ICL showing the electron-dense dotswithin the matrix of layer 2 (L2). Scale bar=150 nm. C. TEM of a section through the epidermis of the trunk revealingthe voluminous cytoplasm of the syncytium (sy). A muscle (mu) can be seen in the blastocoel (bc) as can a portion ofa protonephridium (pr). Both appear enclosed by the basal lamina (bl). Scale bar=400 nm. D. TEM of a section reveal-ing the merging of a membrane-bound vesicle (v) with the overlying ICL. The cytoplasm is largely devoid of organellesexcept for Golgi complex (gc) and apparent ribosomes (dark spots). Scale ba r=500 nm. E. TEM of a section throughthe syncytium (sy) showing invaginations (in) of the plasmalemma at the base of the epidermis. A mitochondrion (mt)is apparent within a membrane-bound vesicle close to the ICL. Scale bar=300 nm. F. TEM of a section revealing abundle of presumed microtubules (mi) within the cytoplasm of the syncytium. Scale bar=500 nm.

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uct of the highly folded intracytoplasmic lamina(ICL) (Figs. 2B–D, 3A–F), which was the electron-dense region at the apical surface of the syncytium.The ICL, in total, consisted of three layers atop thesyncytial cytoplasm. Layer 1 was the most externallayer (cell membrane) and was thin and mostlyelectron dense (Figs. 2C, 3A–F). In some sections,this layer had “peeled” away from layer 2, whichpermitted a better view of its multilaminate substruc-ture (Fig. 3A,D): the apical lamina of layer 1 wasgranular, moderately electron dense, and up to30 nm thick. The underlying lamina of layer 1 wasmore electron dense and 14–16 nm thick. Layer 2was a thickened, mostly homogeneous layer of elec-tron-dense matrix that varied between being amor-phous and somewhat granular (most of the trunk;Figs. 2C, 3) to highly fibrous (just below the cor-ona; Fig. 2D). The amorphous matrix of layer 2 atthe tips of the ridges and just below layer 1 oftencontained electron-dense dots (arrowheads; Fig. 3A,B). In some sections, it was difficult to determine ifthese electron-dense dots were part of layer 1(arrowhead, Fig. 3A) or layer 2 (arrowhead,Fig. 3B). The fibrous regions of layer 2 were onlynoted in highly folded areas of the neck, and somight have been the result of layer 2 being stretchedat these folds. Layer 2 had a consistent thickness ofca. 150–200 nm in the grooves across the trunk, butthickened at the ridges. The grooves were mostlysmooth in appearance, although several containedsmall swellings (probably of layer 1) that were alsoprominent on the sides of the ridges (data notshown) and may correspond to the zipper-likeappearance of the integument when viewed withSEM (see Fig. 2A).

The external ridges prominent in SEM (andappearing as finger-like projections in TEM) werefolds of layers 1 and 2 that formed extensions to700 nm above the surface of the integument. Insome sections, the ridges were bifurcated (Fig. 3F)while others were swollen at their tips (Fig. 3D).These might be artifacts of sectioning or fixation.

Layer 3 was an electron-dense membrane subja-cent to layer 2 and directly on top of the syncytium.This layer was moderately electron dense andappeared finely granular or somewhat fibrous in thetrunk region (see Fig. 3A,B). Anteriorly, this layerwas much more electron dense (Fig. 2C,D). Thick-ness of this layer was highly variable, from 30 to60 nm in most regions of the trunk, but occasionallyup to 120 nm in some sections. Electron-lucentspaces were occasionally present in between layers 2and 3, but the nature of this space (artifact or not)could not be determined.

Syncytium. The syncytium was voluminousthroughout the trunk (1000–3000 nm thick) butthinned out considerably in the neck and coronalregions (compare Figs. 2C and 3C–E). The cyto-plasm was mostly electron lucent and containedrelatively few organelles other than ribosomes,Golgi, and membrane-bound vesicles (Fig. 3D). Thevesicles appeared to be exocytotic because many hadapparently fused with the overlying ICL where theyhad released their contents (Fig. 3D). Several vesi-cles were noted to contain electron-dense materialsand even mitochondria (Fig. 3D,E), while otherswere electron lucent and without any observablecontents (Fig. 2C). Bundles of filaments were pres-ent in scattered positions throughout the syncytiumin the trunk. The bundles contained more than 50hollow filaments, each of which was ca. 23–29 nmin diameter; the bundles were never consistentlyassociated with other organelles, although they werealways positionally close to the basal plasmalemma(10–12 nm thick), which was highly infolded(Fig. 3F). A basal lamina 30–40 nm thick was pres-ent below the plasmalemma and within the invagin-ations of the basal surface (Fig. 3F). The basallamina was considerably thicker in the more ante-rior portions of the trunk where the cytoplasm wasless voluminous (compare Figs. 2C and 3D).Cuticle. The cuticle is defined as the thickened

extracellular matrix (glycocalyx) that serves noskeletal function (see Cl�ement & Wurdak 1991). InS. socialis, the cuticle appeared as a lightly floccu-lent material up to 800 nm thick (Fig. 3A,F). It waspresent in all specimens although it was not consis-tently present in all body regions. It was generallypresent in the trunk and often absent towards theanterior end immediately below the corona.

Discussion

Koehler’s (1965, 1966) seminal research on therotifer integument was important in establishing theunique structure of the intracytoplasmic lamina(ICL) and in showing that it is not, in fact, an extra-cellular cuticle, despite its appearance. He noted thatexocytotic vesicles (hypodermal bulbs of Koehler1965, 1966) are often abundant in the cytoplasmand merge with overlying ICL to release their con-tents, which suggests that the ICL is not extracellu-lar or, in fact, solid, but instead permits movementof vesicles through its matrix. Subsequent studieshave confirmed his findings in a variety of speciesand revealed additional structural variation in theICL (Cl�ement 1969, 1980; Storch & Welsch 1969;Brodie 1970; Schramm 1978; Hendelberg et al. 1979;

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Cl�ement & Wurdak 1991; Ahlrichs 1997). Accordingto Cl�ement & Wurdak (1991), and integrating stud-ies of Ahlrichs (1997), there are three general typesof ICL in rotifers: (1) the bdelloid type, which con-sists a thin external lamina (hypodermal membraneof Koehler 1966) over a thickened internal lamina(dense layer of Koehler 1966); (2) the monogononttype (containing 3 subtypes), which consists of athickened external lamina on top of a thinner basallamina; and (3) the seisonoid type, which consists ofa thin external lamina (sublayer 1 of Ahlrichs 1997)separated from the thicker basal lamina (sublayer IIof Ahlrichs 1997) by a region of cytoplasm.

The microtexture of the ICL in Sinantherina soci-alis is unique as described below, but its ultrastructureis broadly similar to the ICL of other monogononts,consisting of a thin outer membrane (hypodermalmembrane of Koehler 1966), thickened matrix layer,and a relatively thin basal layer. In both cases, thethin apical layer (layer 1 of S. socialis) corresponds tothe outer cell membrane, although the substructure ofthis membrane (or membranes, see Fig. 3A) differsamong the different groups of rotifers includingS. socialis. In S. socialis, this membrane possessed aclear substructure, where there was a finely granularlamina (30 nm thick) atop a thinner electron-denselamina (14–16 nm). Curiously, layer 1 was clearly dif-ferent in substructure and thickness from the basalplasmalemma (10–13 nm), which makes its categori-zation as a standard cell membrane difficult to inter-pret. In two other rotifers, Asplanchna sieboldiLEYDIG 1854 (Monogononta) and Habrotrocha rosaDONNER 1950 (Bdelloidea), the hypodermal mem-brane also shows an unusual substructure: A. sieboldihas five layers reaching 20 nm in thickness (Koehler1965), while H. rosa has a trilaminar membrane17 nm in thickness (Schramm 1978). Both researchersnote that the cellular membranes of their respectivespecies are thicker than typical unit membranes.Unfortunately, there is little qualitative or quantita-tive data on the substructure of cell membrane inother rotifers, so a more detailed comparison is notpermissible.

The more internal and thickened layer of the ICLof S. socialis was either amorphous or finely granular,but always became more fibrous toward the anteriorend. This amorphous or finely granular substructureis similar to what is known for bdelloids (Koehler1966) and some species of Monogononta (subtype 3of Keratella and Trichocerca: Cl�ement & Wurdak1991). Interestingly, in S. socialis, the matrix of layer2 appeared to change toward the anterior end of theanimal, becoming distinctly more fibrous (compareFig. 2C,D). It is unknown if this structural change is

due to stretching of the ICL around folds in the bodywall or if it is always present. It is worth noting thatall sections through the anterior end of five separatespecimens were highly folded. Layer 3 of the ICL inS. socialis was distinct from layer 2 and appearedfinely granular or occasionally fibrous (Fig. 3A,F).Anteriorly, this layer was much more electron densethan in the trunk (compare Figs. 2D, 3). Regardless,this layer appeared grossly similar to the internallayer of other rotifers, but was generally much thinnerbeing only 30–60 nm thick compared to 200 nm inbdelloids (Koehler 1966); quantitative data formonogononts is absent.

Layers 2 and 3 made up the unique ridge-and-groove microtexture of the integument of S. socialis.To date, no other rotifers have been shown to possesssuch a unique microtexture, and while several mono-gononts have spines or thickened ICLs that form alorica and are derived from the external lamina(described in Cl�ement & Wurdak 1991; equivalent tolayer 2 of S. socialis), none create the unusual surfacepatterns seen in S. socialis. It is tempting to hypothe-size that this unique pattern may impart a form oftexture-based deterrent to predators, since the speciesis regularly rejected in studies of predation by fish(Felix et al. 1995) and invertebrates (Walsh et al.2006). In these studies, it is hypothesized that the fourwart-like glands posterior of the corona (asterisks,Fig. 1A) secrete defensive compounds that make therotifers unpalatable, but to date, the nature of thesecretions has not been determined. It is also possiblethat the ridge-and-groove microtexture of the integu-ment functions to capture any secreted fluids from theglands, which may theoretically accumulate withinthe grooves especially if gland secretion is constitutiveand the secretions are not water-soluble.

In addition to the unique ICL of S. socialis, wenote that the syncytium was much more voluminousin this species (up to 3000 nm thick in portions of thetrunk) than in any other described rotifer. In mostspecies, the cytoplasm is extremely thin and less than100 nm thick (Koehler 1965, 1966; Schramm 1978),although it reaches 1000 nm in preoral and caudalregions of H. rosa (Bdelloidea) (Schramm 1978). Wehypothesize that the voluminous cytoplasm in thetrunk of S. socialis acts synergistically with the ICLto provide skeletal support to animals that are regu-larly “upright” in posture as they feed and interactwith other colony members. To this end, the bundlesof filaments that are sparsely distributed throughoutthe cytoplasm might also make a skeletal contribu-tion, though we have few details on the exactposition, abundance, or lengths of these filaments.Longitudinal sections through these bundles were

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never observed, but the diameter of individualfilaments (23–29 nm) roughly corresponds to thediameter of microtubules present in other inverte-brates (de-Th�e 1964; Chalfie & Thomson 1979),hence, our interpretation of the filaments as microtu-bules. We do note that the general appearance andregular arrangement of the filaments is similar toother types of cell inclusions such as protein crystal-loids (Threadgold 1965; Reger 1969), but their hollowsubstructure is much more similar to microtubules.To date, the only other rotifer known to possess bun-dles of microtubules in its epidermis is the marineectosymbiont Seison nebaliae GRUBE 1861 (Ahlrichs1997). In this species, the microtubules are also pres-ent in the trunk epidermis (S. nebaliae has a cellularepidermis), but their substructure was not examined,thus their precise identity remains to be determined.

We have demonstrated that the fine structure ofS. socialis is unique among previously examined rot-ifers, but without further details on other species ofGnesiotrocha, we hesitate to speculate on whetherthe integument and its peculiarities (e.g., microtexture,cytoplasmic volume, microtubules) are truly excep-tional within Rotifera. We expect that with furtherexamination of gnesiotrochans—taking into accounttheir varied morphologies and lifestyles—the charac-teristics described here will be found in other speciesand may therefore be useful in defining phylogeneticrelationships (e.g., as noted by Cl�ement & Wurdak1991); alternatively, they may correlate with particu-lar lifestyles (e.g., sessility, coloniality) suggestingconvergence.

Acknowledgments. This research was supported by theNational Science Foundation (DEB 1257110 to RH). Anyopinions, findings, and conclusions or recommendationsexpressed in this material are those of the authors and donot necessarily reflect the views of the National ScienceFoundation.

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