Ultrastructure of the Spermatozoon of Leiopelma Hochstetteri (Amphibia, Anura, Leiopelmatidae)
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Ultrastructure of the spermatozoon of Leiopelma hochstetteri (Amphibia, Anura, Leiopelmatidae) David M. SCHELTINGA Barrie G. M. JAMIESON Department of Zoology and Entomology, University of Queensland, Brisbane, Qld, 4072 (Australia) [email protected] Karen E. EGGERS Ecology, Institute of Natural Resources (INR), Massey University, Private Bag 11222, Palmerston North (New Zealand) David M. GREEN Redpath Museum, McGill University, 859 Sherbrooke St. West, Montreal, Quebec H3A 2K6 (Canada) Scheltinga D. M., Jamieson B. G. M., Eggers K. E. & Green D. M. 2001. — Ultrastructure of the spermatozoon of Leiopelma hochstetteri (Amphibia, Anura, Leiopelmatidae). Zoosys- tema 23 (1) : 157-171. ABSTRACT The spermatozoon of the basal frog Leiopelma hochstetteri Fitzinger, 1861 is ex- amined and compared with previously investigated lissamphibian spermatozoa. L. hochstetteri spermatozoa share with those of urodeles and Ascaphus the ple- siomorphic (primitive) features of an elongate conical acrosome, and an elongate nucleus, with distinct nuclear shoulders, which tapers to a point (as the nuclear rostrum) in the subacrosomal space. The spermatozoon of L. hochstetteri, alone among the Anura, resembles those of urodeles in having a juxta-axonemal fibre at doublet 8. L. hochstetteri shares with Ascaphus truei (homoplastically) the reduction of the undulating membrane to a short, thick paraxonemal rod. L. hochstetteri spermatozoa have the plesiomorphic condition, also seen in dip- noans, of a cytoplasmic collar, containing mitochondria, around the axoneme. Although the sperm of L. hochstetteri lacks the endonuclear perforatorium seen in Ascaphus, Bombinidae, urodeles and caecilians, it has what may be remnants of an endonuclear perforatorium within the nuclear rostrum. No synapomor- phic spermatozoal characters are present to support the placement of Leiopelma KEY WORDS Amphibia, and Ascaphus in the same family. Spermatologically, Leiopelma is the Anura, plesiomorph sister taxon of all other anurans and is thus confirmed as the most Leiopelmatidae, basal (primitive) anuran. A further finding is that the Pelodytidae, sometimes spermatozoa, ultrastructure, considered basal archaeobatrachians, share the synapomorphy of a conical phylogeny. perforatorium divided into fibres with the Bufonoidea. ZOOSYSTEMA • 2001 • 23 (1) © Publications Scientifiques du Muséum national d’Histoire naturelle, Paris. www.mnhn.fr/publication/ 157 Scheltinga D. M., Jamieson B. G. M., Eggers K. E. & Green D. M. RÉSUMÉ Ultrastructure du spermatozoïde de Leiopelma hochstetteri (Amphibia, Anura, Leiopelmatidae). Le spermatozoïde de la grenouille primitive Leiopelma hochstetteri Fitzinger, 1861est examiné et comparé aux spermatozoïdes de lissamphibiens précédem- ment étudiés. Le spermatozoïde de L. hochstetteri partage avec les Urodèles et Ascaphus les caractéristiques plésiomorphes (primitives) suivantes : un acro- some conique et allongé, un noyau allongé, avec des épaules nucléaires dis- tinctes, et qui s’effile dans l’espace subacrosomal, formant un rostre nucléaire. Le spermatozoïde de L. hochstetteri est le seul parmi les Anoures à ressembler à ceux des Urodèles par une fibre juxta-axonémale associée au doublet 8. L. hochstetteri a en commun avec Ascaphus truei (de manière homoplasique) la réduction de la membrane ondulante en une baguette paraxonémale courte et épaisse. Les spermatozoïdes de L. hochstetteri ont l’état de caractère plésiomorphe, également rencontré chez les Dipneustes, d’un collier cytoplasmique conte- nant des mitochondries, autour de l’axonème. Bien que le spermatozoïde de L. hochstetteri ne possède pas le perforatorium endonucléaire trouvé chez Ascaphus, les Bombinidae, les Urodèles et les Caeciliens, il présente ce qui peut être les restes d’un perforatorium endonucléaire dans le rostre nucléaire. Aucune synapomorphie spermatique ne soutient l’inclusion de Leiopelma et MOTS CLÉS Amphibia, d’Ascaphus dans la même famille. Du point de vue spermatologique, Leiopelma Anura, est le taxon-frère plésiomorphe de tous les autres Anoures, ce qui confirme sa Leiopelmatidae, position d’Anoure le plus basal. On remarque aussi que les Pelodytidae, par- spermatozoïdes, ultrastructure, fois considérés comme des Archaeobatraciens basaux, partagent la synapo- phylogénie. morphie d’un perforatorium conique divisé en fibres avec les Bufonoidea. INTRODUCTION As observed in captive Leiopelma hochstetteri, amplexus is inguinal and occurs on land, in shal- Frogs of the genus Leiopelma Fitzinger, 1861 are low water, or at the waters edge between the endemic to New Zealand and are considered to months of October and May (Bell 1978). Breed- be among the most primitive (basal) of all living ing sites are generally under rocks and logs in and anurans (Cannatella 1985; Green & Cannatella around water seepages and smaller streams, where 1993; Duellman & Trueb 1994). Leiopelma clutches of 10 to 22 eggs are laid and from which hochstetteri Fitzinger, 1861 is a semi-aquatic, tailed swimming larvae hatch (Bell 1982, 1985). highly cryptic and sedentary frog which lives and Males may occur in association with egg clutches breeds in scattered remnant populations in moist but do not actively incubate the eggs, unlike seepages and along the edges of forest streams on other species of this genus. Furthermore, the lar- the North Island and Great Barrier Island of New vae differ from their congeners in that they do Zealand (Bell 1978; Tessier et al. 1991). They not metamorphose on the back of a resident male grow to a maximum known snout-vent length of frog, but metamorphose post-hatching and dis- 46 mm (Mclennan 1985; Slaven 1992) in close perse soon after (Bell 1985). agreement with lengths of 50 mm in subfossil Among the extant Anura only Ascaphus Stejneger, deposits (Worthy 1987). They are mostly dull 1899 and Leiopelma retain the plesiomorphic char- brown frogs but dark green morphs also occur acters of nine presacral amphicoelous vertebrae and (Barwick 1961; Whitaker 1996). “tail-wagging” muscles. Although these are sym- 158 ZOOSYSTEMA • 2001 • 23 (1) Spermatozoon of Leiopelma (Amphibia, Anura, Leiopelmatidae) plesiomorphies, Ascaphus and Leiopelma have been Zealand on June 29th 1998 were enclosed in united in a single family, the Leiopelmatidae, 10 cm × 6 cm plastic containers lined with damp together with the extinct Patagonian genera tissue paper. Each container was then placed Notobatrachus Reig, 1957 and Vieraella Reig, 1961 within a 20 cm × 30 cm polystyrene container, (Duellman & Trueb 1994). However, Green & maintained at 10 oC for transport to Massey Cannatella (1993) concluded that, as Ascaphus and University. Frogs were maintained on a diet of Leiopelma share no synapomorphic characters, crickets and wax moth larvae for one week they should be placed in separate families, before transport to Victoria University. At Ascaphidae and Leiopelmatidae respectively. Victoria University each frog was euthanased Other research into the phylogenetic relationships with Benzocaine. The testes from the only male of these basal anuran families has also produced frog collected, svl = 34.4 mm, (FT4071, conflicting conclusions. Cannatella (1985) on the C. Daugherty field collection number, Victoria basis of morphological data considered Ascaphus University), were fixed for transmission electron truei Stejneger, 1899 to be the sister taxon of all microscopy (TEM) in 3% glutaraldehyde in other anurans (see also a reanalysis of Cannatella’s 0.1M sodium phosphate buffer (pH 7.2) at 4 oC data by Hillis 1991), whereas, from an analysis for 2 h, being agitated for the first hour. Testis of isozyme loci, Green et al. (1989) considered material was then posted to Brisbane, Australia, Leiopelma to be the probable sister group of all at ambient temperature for TEM processing. other frogs. In contrast to the above views, an The testicular tissue was diced into 1-2 mm3 extensive study of amphibian 12S and 16S rRNA portions and then rinsed in 0.1M phosphate data by Hay et al. (1995) indicated that the buffer, post-fixed for 80 min in similarly- two anuran suborders Archaeobatrachia and buffered 1% osmium tetroxide, rinsed in buffer, Neobatrachia are each monophyletic. That dehydrated through an ascending ethanol series, Ascaphus and Leiopelma are sister groups within and infiltrated and embedded in Spurr’s epoxy the Archaeobatrachia, with the most basal lineage resin. Sections were cut with diamond knives, of the Archaeobatrachia being the Pelobatoidea on a LKB 2128 UM IV microtome. Thin sec- (Pelodytidae + Pelobatidae). While Hay et al. tions, 50-80 nm in thickness, were collected on (1995) found Ascaphus and Leiopelma to be sister carbon stabilized, colloidin-coated, 200 µm groups, this needs not necessitate placing them in mesh copper grids, rinsed in distilled water, the same family, the Leiopelmatidae, as suggested stained for one minute in Reynold’s lead citrate, by them. rinsed in distilled water, then placed in 6% In this study we attempt to clarify the confused aqueous uranyl acetate for six minutes, rinsed phylogenetic relationships of Leiopelma hochstet- again, and stained for a further three minutes in teri to other families of frogs by comparing its lead citrate before final rinsing. Electron micro- spermatozoal ultrastructure with that of pre- graphs were taken on an Hitachi 300 electron viously examined lissamphibian spermatozoa, in microscope at 75 kV. particular that of Ascaphus truei. Light microscopic observations of spermatozoa from glutaraldehyde-fixed tissue squashes,