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ULTRASTRUCTURE OF THE SPERMATOZOA OF LITORIA LONGIROSTRIS(HYLIDAE, ANURA, AMPHIBIA): MODIFICATIONS FOR PENETRATION OF A

GELATINOUS LAYER SURROUNDING THE ARBOREAL EGG CLUTCH

DAVID M. SCHELTINGA, BARRIE G M. JAMIESON AND KEITH R. MCDONALD

Scheltinga, D.M., Jamieson, B.G.M. & McDonald, K.R. 2002 5 31: Ultrastnicture of thespermatozoa of Litoria longirostris (Hylidae, Anura, Amphibia): modifications forpenetration of a gelatinous layer surrounding the arboreal egg clutch. Memoirs of theQueensland Museum 48(1): 215-220. Brisbane. ISSN 0079 8835.

Spermatozoa of Litoria longirostris are highly modified for its unique mode of fertilisationand differ distinctively from the sperm of the 10 Litoria species previously examined. L.longirostris spermatozoa are longer in head, tail and total. The head is long and straight with adistinct acrosome vesicle compared to a short curved head in other Litoria spermatozoa. Theacrosome vesicle is well developed and surrounds approximately the apical third of theperforatorium only. The perforatorium is a solid homogenous rod that attaches to the nucleusasymmetrically along one side. The nucleus and midpiece are similar in size and structure tothose of other Litoria species. The axial fibre is greatly enlarged and a juxta-axonemal fibreat doublet 3, usual in anuran sperm, is absent.

Although the spermatozoa of L. longirostris are highly modified, having secondarily lost thebufonoid synapomorphy of a putative conical perforatorium consisting of fibres, it can stillbe distinguished as a eubufonoid by the mitochondrial collar. Thus, we assert that spermmorphology is correlated with phylogenetic relationships as well as mode of fertilisation andthat spermatozoal morphology can provide useful information in resolving phylogeneticrelationships. El Frog, spermatozoa, ultrastructure, fertilisation, Litoria longirostris.

D.M. Scheltinga and B.G.M. Jamieson, Department of Zoology and Entomology, Universityof Queensland, Brisbane, Qld, Australia, 4072; K.R. McDonald, Queensland Parks andWildlife Service, PO Box 834, Atherton, Qld, 4883, Australia; received 10 April 2002.

Litoria longirostris is a relatively small (ca25mm) grey-brown frog inhabiting the graniteupland, rainforest streams of the McIlwraithRange, Cape York Peninsula, Queensland. It isunique amongst Australian frogs in itsreproductive mode of depositing egg clutches onvegetation and rocks overhanging water bodiesin rainforest streams (McDonald & Storch,1993). The lime green eggs are large (2.0-2.4mmdiameter) and surrounded by a 6mm thickgelatinous capsule. Tadpoles in the clutch aresimilar in colour to the eggs during development,before turning a pale brown on the dorsal surfaceprior to hatching. Parental care has been observedwith adult frogs straddling the clutch (McDonald& Storch, 1993). The exact method of amplexusand fertilisation remains unknown.

Sperm ultrastructure has previously beenexamined in 10 species of the Australian hylidLitoria: aurea, caerulea, eucnemis, fallax,gracilenta, lesueuri, moorei, peronii, rheocolaand rubella (Lee & Jamieson, 1993; Meyer et al.,1997; Jamieson, 1999). Although these frogsreproduce in a wide variety of habitats, their

sperm are similar. With regard to correlation ofreproductive mode with sperm structure, Garridoet al. (1989) noted that complex flagella occur inspecies in which fertilisation occurs in a non-aquatic environment and in which developmentmay be viviparous, direct, or aquatic. In ter-restrial breeders, notably some of the foam-nesting rhacophorids, the spermatozoon reachesits highest degree of modification. Male Rhaco-phorus arboreus, R. schlegelii and Chiromantisxerampelina, which shed their sperm onto a foamnest, have highly modified "corkscrew shaped"sperm (Oka, 1980; Mainoya, 1981; Mizuhira etal., 1986; Wilson et al., 1991; Jamieson, 1999).However, in some foam-nesting rhacophorids, aswell as those like Buergeria buergeri which donot build a foam-nest but lay their eggs instreams, the spermatozoa have a long head andthin tail, neither of which is spiral (Fukuyama etal., 1993; Kuramoto, 1996; Kuramoto & Joshy,2000, 2001).

The 'arboreal-nesting' reproductive method ofL. longirostris is similar to that of several speciesfrom divergent anuran families (Duellman &

216^MEMOIRS OF THE QUEENSLAND MUSEUM

LITORIA LONGIROSTRIS SPERMATOZOON^217

Trueb, 1986) and thus allows correlation betweenterrestrial nesting and sperm ultrastructure to befurther examined. We here give the first descript-ion of the spermatozoon of L. longirostris.

MATERIALS AND METHODS

Three male L. longirostris Tyler & Davies,1977 were collected from near egg clutches atUpper Peach Creek, McIlwraith Range, NorthQueensland, on 26-27 September 1995(QMJ62099, J62100, J62102). The frogs werekilled with a lethal injection of sodiumpentobarbital shortly after capture. The testeswere quickly removed and fixed for transmissionelectron microscopy (TEM) in 3%glutaraldehyde in 0.1M sodium phosphate buffer(pH 7.2) at 4°C for at least two hours before beingtransported at ambient temperature to Brisbanefor processing and sectioning. On arrival inBrisbane, material for TEM examination wasdiced into 1 mm 3 pieces and rinsed in 0.1Msodium phosphate buffer; post-fixed for 80 minin similarly buffered 1% osmium tetroxide;rinsed in buffer; dehydrated through anascending ethanol series; and infiltrated andembedded in Spurr's epoxy resin (Spurr, 1969).

Sections were cut with a diamond knife on aLIKE 2128 UM IV microtome. Thin sections,50-80nm thick, were collected on carbonstabilised, colloidin-coated, 200um mesh coppergrids, stained for 30 s in Reynold's lead citrate(Reynolds, 1963), rinsed in distilled water, thenplaced in 6% aqueous uranyl acetate for 4 min,rinsed in distilled water, and stained for a further2 min in lead citrate before final rinsing. Electronmicrographs were taken on an Hitachi 300electron microscope at 75kV and a JEOL 100-selectron microscope at 60kV.

A drop of glutaraldehyde-fixed spermatozoa,for examination by scanning electron

microscopy (SEM), was placed on a clean smallround cover slip. Dehydration of the spermatozoathrough an ascending ethanol series and finalamyl acetate was achieved by adding a drop ofeach reagent in turn, after removal of the previousfluid with filter paper. The spermatozoa weresubjected to critical point drying, gold sputtercoated, and photographed on a JEOL 6400 SEM.

Photographs of spermatozoa, from glutaral-dehyde-fixed tissue squashes, were made usingan Olympus BH2 microscope with Nomarskiinterference contrast and attached 0M-2 camera.

RESULTS

The testicular spermatozoa of L. longirostrisare filiform and average 82.7um (n-20, SD-3.3)in length (range 74.1-87.4m). The sperm-atozoon is composed of a straight head region(acrosome complex and nucleus) 34.211m long (n=9, SD=1.9), a midpiece 2.49um long (n=5,SD-0.32), and a tail 46.3 um long (n=11, SD=1.7) (Fig. 1J). Under light microscopy a distinctacrosome vesicle, perforatorium, nucleus,midpiece, axial fibre, undulating membrane andaxoneme can be clearly seen.

ACROSOME COMPLEX. The acrosomecomplex is 24.6um long (n=6, SD=1.1) andcomposed of an elongate cylindrical acrosomevesicles surrounding the apical portion of theputative perforatorium (Fig. 1A,J). The acrosomevesicle is 10.4um long (n=5, SD=0.08),membrane bound and filled with moderatelyelectron-dense material (Fig. 1A,B). Theperforatorium continues posteriorly without anyassociated structures for some distance beforeattaching asymmetrically to one side of thenucleus (Fig. 1C,D,H). The perforatorium is asingle unit, not divided into separate sheaves/fibres, with a constant diameter throughout its

FIG. 1. Litoria longirostris. A-I, TEM. A, Longitudinal section (L.S.) of the apical region of a spermatozoon,showing the acrosome vesicle surrounding only the anterior region of the perforatorium; B-G, Successivetransverse sections (T.Ss) through the spermatozoon as indicated; B, through the apical region of the acrosomecomplex; C, through the perforatorium; D, through the junction of the perforatorium and nucleus; E, through theposterior region of the nucleus; F, through the distal centriole; G, through the midpiece; H, L. S. of the nucleus(showing the perforatorium lying along one side of the nucleus, see also D) and midpiece (showing themitochondrial collar); I, T.S. of the tail. Note the absence of a juxta-axonemal fibre at doublet 3 and therelatively large axial fibre (see also F and G); J, Whole testicular spermatozoon shown by SEM, showing thehead (acrosome vesicle, perforatorium and nucleus), midpiece and tail (axial fibre, undulating membrane andaxoneme). B-G and Ito the same scale as indicated. A, H and J scales as indicated. Abbreviations: a = axoneme;af = axial fibre, av = acrosome vesicle; cc = cytoplasmic canal; cy = cytoplasm; dc = distal centriole; e =endpiece; f= nuclear fossa; g = putative glycogen granules; m = mitochondrion; mc = mitochondrial collar; mp= midpiece; n = nucleus; p = perforatorium; pc = proximal centriole; pcm = pericentriolar material; um —undulating membrane.

nuclear membrane

putative glycogen

nucleus

vacuole

plasma membrane

nuclear fosse

_^ proximal centnole

1:1-17 . Mb: \ pencentnolar material

distal centriole

cytoplasm

cytoplasmic canal

mitochondrion

m tochondrial collar

1 p.m

a'nemel

^ undulating membrane

axial fibre

FIG. 2. Drawing of a longitudinal section through the neck region of aspermatozoon of L. longirostris. Drawn from a TEM micrograph.

218^MEMOIRS OF THE QUEENSLAND MUSEUM

length, is moderately electron-dense and is notbounded by a membrane.

NUCLEUS. The nucleus is 11.07um long (n=2,SD=0.09), cylindrical, and electron-dense(condensed chromatin) (Fig. 1E,H). Anteriorly,the nucleus tapers to a distinct point alongside theperforatorium (Fig. 1H). Distinct nuclearshoulders are absent. At the base of the nucleus awell-developed nuclear fossa is present (Figs 1H;2). At this level the nucleus is 1.07jtm in diameter(n = 8, SD = 0.05).

NECK/MIDPIECE. Within the basal nuclearfossa lies the proximal centriole which issurrounded by pericentriolar material thatconnects it to the nuclear fossa, distal centrioleand the axial fibre (Figs 1F,H, 2). The proximalcentriole lies at —75° to the long axis of the

nucleus and at right angle to the distalcentriole which forms the basal bodyof the axoneme (Fig. 2). Eachcentriole is composed of 9, circularlyarranged, triplets of shortmicrotubules. The axial fibre extendsthrough the neck region to the levelof the base of the nucleus (Figs 1H;2). A short mitochondrial collar,containing scattered mitochondria,vacuoles and putative glycogengranules, surrounds the anteriorportion of the tail but is separatedfrom it by a gap, the cytoplasmiccanal (Figs 1F-H, 2).

TAIL COMPLEX. The tail complex(Figs 1I,J, 2) is composed of a 9+2axoneme and an axial fibre, the fibrebeing connected by a thin undulatingmembrane to doublet 3. Ajuxta-axonemal fibre at 3 is absent.The axial fibre is well-developed andcircular in transverse sectionthroughout its length. Anteriorly, it isenlarged, being — 0.95 pm in diameter(Fig. 1F-I). It continues posteriorly,decreasing in diameter, for almost theentire length of the tail resulting inonly a very short portion of theaxoneme extending as an endpiece(Fig. 1J).

DISCUSSIONSpermatozoa of all 10 species of

Litoria which have previously beenexamined (Lee & Jamieson, 1993,Meyer et al., 1997; Jamieson, 1999)

are similar to each other. All closely resembleBafo sperm and exhibit the bufonoidsynapomorphy of a conical perforatorium ofseparate sheaves and the eubufonoid synapo-morphy of a long mitochondrial collar separatedfrom the tail by a cytoplasmic canal (Pugin-Rios,1980; Lee & Kwon, 1992; Lee & Jamieson, 1993;Kwon & Lee, 1995). Spermatozoa of L. long-irostris differ from these Litoria species. How-ever, despite these differences the spermatozoashow characters which are synapomorphic forthe eubufonoids.

Spermatozoa of L. longirostris are easilydistinguished from those of the 10 Litoria speciespreviously examined. Litoria longirostrisspermatozoa are longer, 83)1m, compared with47-56)tm in the 6 Litoria species examined by

LITORIA LONGIROSTRIS SPERMATOZOON^219

Lee & Jamieson (1993), and the length of thesperm head is approximately twice the length of14-18iim recorded in the latter species. The headis long and straight with a distinctwell-developed acrosome vesicle, contrastingwith a short curved head in the other investigatedLitoria spermatozoa. The acrosome complex ishighly modified relative to the other species. Inthe latter, a thin walled conical acrosome vesiclecompletely caps a putative conical perforatoriumof separate sheaves of perforatorial material andthe acrosome complex symmetrically caps thetapered point of the nucleus. In L. longirostris,the acrosome vesicle differs in surrounding onlythe apical third, approximately, of theperforatorium. The perforatorium differs inbeing a solid homogenous cylinder that attachesto the nucleus asymmetrically along one side.Most, if not all, of the increase in length of thehead in L. longirostris is due to the great length ofits acrosome complex. It is deduced that L.longirostris spermatozoa have secondarily lostthe bufonoid synapomorphy of a conicalperforatorium consisting of fibres.

In some foam-nesting rhacophorids the spermhead shows modifications different from those ofL. longirostris and forms a spiral (Oka, 1980;Mainoya, 1981). It is unknown if there are anyultrastructural modifications to the sperm ofthose foam-nesting rhacophorids that do notshow a spiral head.

In view of the fact that the spermatozoa of the10 previously examined Litoria species havesuch uniform structure despite differingreproductive modes (lotic vs lentic nesting). Itseems likely that the acrosomal modificationsobserved in L. longirostris are an adaptation topenetration of the thick gelatinous masssurrounding the large eggs.

The nucleus and midpiece of L . longirostris aresimilar in size and structure to those Litoriaspecies previously examined and show theeubufonoid synapomorphy of a mitochondrialcollar separated from the tail by a cytoplasmiccanal. The tail of L. longirostris spermatozoadiffers in its length (46um compared to 35-40um(Lee & Jamieson, 1993)), having a greatlyenlarged axial fibre and in the absence of ajuxta-axonemal fibre at doublet 3. All previouslyexamined Litoria spermatozoa have ajuxta-axonemal fibre at doublet 3. Lee &Jamieson (1993) proposed that the enlargementof the juxta-axonemal fibre observed in L. fallax,L. gracilenta and L. lesueuri was a weak

synapomorphy uniting them. It appears that thepresence and size of the juxta-axonemal fibremay provide a useful character in the ex-amination of phylogenetic relationships betweenspecies of Litoria. Conversely, loss of thejuxta-axonemal fibre may not be correlated withphylogenetic relationships and may be anadaptation to the unique fertilisation biology ofL.longirostris.

The spermatozoa of L. longirostris differgreatly from those of foam-nesting rhacophoridsthat have been examined ultrastructurally in tail,in addition to acrosomal, characters. In theserhacophorids the sperm possess two axonemeswhich are surrounded by microtubules in apseudocrystalline matrix. Furthermore,juxta-axonemal fibres, axial fibre and undulatingmembrane are all absent (Mainoya, 1981;Mizuhira et al., 1986; Wilson et al., 1991;Jamieson, 1999). The loss of a juxta-axonemalfibre, axial fibre and undulating membraneappear each to be a synapomorphy of theRanoidea, though these states are questionablyindependent of each other. The only notablesimilarities, albeit superficial, between thespermatozoa of L. longirostris and foam-nestingrhacophorids are the asymmetrical attachment ofthe acrosome complex to the nucleus and theabsence of a juxta-axonemal fibre associatedwith doublet 3. On present evidence it appearsthat these similarities are homoplasies.

The Mexican hylid Pachymedusa dacnicolorhas a similar reproductive mode (Bagnara et al.,1986) to L. longirostris and is the only otherarboreal-nesting frog to have had its spermatozoaexamined ultrastructurally (Rastogi et al., 1988).P. dacnicolor spermatozoa are very similar tothose of the 10 aquatic-nesting Litoria speciespreviously examined and thus differ from L.longirostris in its acrosome vesicle whichcompletely caps a conical perforatoriumcomposed of fibres and a juxta-axonemal fibre atdoublet 3. There is one similarity between thespermatozoa of these 2 arboreal-nesting hylidsthat may reflect their shared fertilisation biologyand that is the thick axial fibre. In both P.dacnicolor and L. longirostris the axial fibre isapproximately lum in diameter compared to adiameter of approximately 0.2um in the otherLitoria examined. However, within the widerAnura, differences in the sperm tail appear morereflective of phylogeny than fertilisation biology(van der Horst et al., 1995; Meyer et al., 1997).

Although the spermatozoon of L. longirostris

220^MEMOIRS OF THE QUEENSLAND MUSEUM

is highly modified for its unique fertilisationbiology it can still be distinguished as that of aeubufonoid by the mitochondrial collar. Thus, weagree with Garrido et al. (1989) that amphibiansperm morphology is correlated with broadphylogenetic relationships (here at thesuprafamilial, eubufonoid level) as well as themode of fertilisation. Therefore, spermatozoonmorphology can provide useful information inresolving phylogenetic relationships at varioustaxonomic levels despite unique fertilisation bio-logy resulting in highly modified spermatozoa.

ACKNOWLEDGEMENTS

Lina Daddow is thanked for excellent technicalassistance with aspects of SEM. Wayne E. Martin(QPWS) and Jeanette A. Covacevich (Queens-land Museum) are thanked for assisting withcollection of the frogs. This work was madepossible by a Departmental Research Grant toDMS and an Australian Research Council grantto BGMJ. Illustrations are by DMS. Theconstructive comments of an anonymousreviewer are gratefully acknowledged.

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