MURDOCH RESEARCH REPOSITORY This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination. The definitive version is available at http://www.tandfonline.com/doi/pdf/10.1080/13235818.2013.782791 Klunzinger, M.W., Beatty, S.J., Morgan, D.L., Thomson, G.J. and Lymbery, A.J. (2013) Morphological and morphometrical description of the glochidia of Westralunio carteri Iredale, 1934 (Bivalvia: Unionoida:Hyriidae). Molluscan Research , 33 (2). pp. 104-109. http://researchrepository.murdoch.edu.au/13481/ Copyright: © 2013 The Malacological Society of Australasia and the Society for the Study of Molluscan Diversity It is posted here for your personal use. No further distribution is permitted. Morphological and morphometrical description of the glochidia of Westralunio carteri Iredale, 1934 (Bivalvia: Unionoida:Hyriidae) MICHAEL W. KLUNZINGER*, STEPHEN J. BEATTY, DAVID L. MORGAN, GORDON J. THOMSON & ALAN J. LYMBERY Freshwater Fish Research, School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150 Australia. *Corresponding author Email: [email protected] Abstract Most freshwater mussel (Bivalvia: Unionoida) larvae (glochidia in Margaritiferidae, Hyriidae and Unionidae) are fish parasites. Knowledge of the larval morphology and the mechanism of release in freshwater mussels is useful in species systematics and ecology. Westralunio carteri Iredale, 1934 is the only unionoid from south-western Australia. Little information is available on its biology and its glochidia have never been described. The aim of this study was to describe the glochidia of W. carteri and method of their release. Glochidia within vitelline membranes were embedded in mucus which extruded from exhalent siphons of females during spring/summer; they then hatched from vitelline membranes but remained tethered by a larval thread and began characteristically ‘winking’. Shells (n = 120) were subtriangular, 308 µm long (±0.83 SE), 251µm high (±0.73 SE) and had a hinge length of 212µm (±0.78 SE). Larval teeth were singular with interlocking cusps and convex or concave basal protuberances on opposing valves. Running head: Glochidia of Westralunio carteri Key words: Larval tooth, functional anatomy, Carter’s freshwater mussel, Velesunioninae, Palaeoheterodonta 1 Introduction Freshwater mussels (Bivalvia: Unionoida) are a widely distributed order of palaeoheterodonts inhabiting lotic and lentic freshwater habitats on every continent apart from Antarctica with an estimated 796 (Bogan & Roe 2008) to 854 species (Graf & Cummings 2006). Most freshwater mussels have a larval stage (called glochidia in the families Margaritiferidae, Unionidae and Hyriidae) that is parasitic on fishes, and sometimes amphibians (Bauer &Wächtler 2001; Strayer 2008). Glochidia morphology is variable within and between the various taxonomic groups (Surber 1912; Wächtler et al. 2001; Sayenko 2006). Unionoida in the Australasian region (Australia, New Guinea, New Zealand and the Solomon Islands) is mostly comprised of the family Hyriidae, although New Guinea also contains two representatives provisionally accepted as Unionidae (Walker et al. 2001). The last systematic review of the Australasian Hyriidae (McMichael & Hiscock 1958) was based primarily on the shell and anatomy of adults. This phylogeny is uncertain, however, because taxa beneath the family level are defined by morphological features which vary in response to local environmental conditions (Balla & Walker 1991; Walker et al. 2001; Baker et al. 2004), soft anatomy lacks differentiation and morphological descriptions of glochidia are incomplete (Ponder & Bayer 2004; Walker 2004). Although descriptions of the glochidia of the Australasian Hyriidae are fragmentary, diversity in larval tooth morphology, shell shape and shell dimensions suggest that they may have systematic value (Walker et al. 2001). Of the 30 nominal species in nine genera of Australasian Hyriidae, glochidia have been described for 10 species (Percival 1931; Parodiz & Bonetto 1963; Walker 1981; Humphrey 1984; Jones et al. 1986; Jupiter & Byrne 1997; Walker et al. 2001). Glochidia release strategies have received a lot of attention in many Northern Hemisphere species (e.g. Kat 1984; Kraemer & Swanson 1985; Haag et al. 1995; Barnhart et al. 2008), but little is known about the mechanism of release in the Australasian Hyriidae (but see Atkins 1979; Jones et al. 1986). Walker et al. (2001) suggest that glochidial morphology may have taxonomic potential in the Australasian Hyriidae, noting that reproductive characters and 2 glochidial morphology have long been used in South American hyriid systematics (see Pimpão et al. 2012). Westralunio carteri Iredale, 1934 is the only member of the genus to reside in Australia (McMichael &Hiscock 1958; Walker et al. 2001). The species is restricted to south- western Australia with a range extending from the Moore River catchment southward and eastward to the Kent River and has two outlying populations in the Goodga and Waychinicup Rivers (Morgan et al. 2011; Klunzinger et al. 2012a). Despite having been accepted as a unique taxon for nearly 80 years, the glochidia of W. carteri have never been described. In this paper, we describe glochidial morphology and shell dimensions and illustrate the release of glochidia by W. carteri for the first time. This information will contribute to the current state of knowledge of the Hyriidae and provide data potentially useful in future systematic assessments of the Hyriidae. Materials and methods Gravid female W. carteri (n = 3 each) were collected from two populations, separated by a distance of approximately 200 km: one in the Canning River (32°07΄S; 116°01΄E) and the other from the Collie River (33°38΄S; 115°91΄E). Live mussels were hand-collected and transported live to the laboratory in plastic buckets containing river water. Samples were maintained in the buckets at room temperature, observed for glochidia release and subsequently dissected. Gravidity was apparent macroscopically from the appearance of thickened, orange to reddish coloured marsupia in the inner two thirds of the inner demibranchs of the females’ gills (Walker 1981; Jones et al. 1986; Byrne 1998). Glochidia were removed using a pipette, placed on a glass slide and examined under a light microscope for behaviour. A sub-sample of live glochidia were removed for scanning electron microscopy (SEM) and gills containing mature glochidia were preserved in 100% ethanol for later analysis. A total of 120 preserved mature glochidia (n = 20 from each of three females within each locality) were flushed from the females’ gills with distilled water and placed on a glass slide. Intact mature glochidia, free of their vitelline membrane, were measured to the nearest 10 µm for length, height and hinge length (Figure 1) using a graticule ruler within the eyepiece of a MoticBA-210 light microscope. For SEM, live glochidia were fixed for 2h in 3 gluteraldehyde, dehydrated in graded ethanols, placed on a glass cover slip lined with double- sided sticky tape attached to a specimen stub, critical point dried, sputter-coated with gold, and examined and photographed with a Philips XL 20 SEM. Given that terminology used to describe glochidia characteristics varies in the literature (Pimpão et al. 2012), some definitions of terms are required here. ‘Glochidial’ or ‘larval’ tooth is the distal projection proximal to the ventral apex to either valve projecting inward or more-or-less perpendicular to the lateral plane of either valve of the glochidia (Atkins 1979; Mansur & Silva 1999; Morgan et al. 2011; Klunzinger 2011; Klunzinger et al. 2011; 2012b). ‘Cusps’ are sharp terminations of the glochidial teeth (Mansur & Silva 1999; Pimpão et al. 2012). A ‘protuberance’ refers to a rounded projection originating from the base of the glochidial/larval tooth (Walker 1981; Vale et al. 2005; Pimpão et al. 2012). The ‘larval thread’ is a strand of tissue inserted between the hinge of the valves which unravels and remains attached to the vitelline membrane upon glochidial ‘hatching’ (Matteson 1948; Hiscock 1951; Atkins 1979; Jones et al. 1986; Jupiter & Byrne 1997). <FIG 1 ABOUT HERE> Results Female W. carteri released thick strands of mucus that contained glochidia enveloped in vitelline membranes. Upon release, the mucus strands began to thin, liberating mature glochidia still within their vitelline membranes. Shortly thereafter (ca. 1 min), glochidia excised themselves by expanding their valves, rupturing their vitelline membranes (Figure 2). The larval thread then unraveled, still attached to the vitelline membrane and the soft tissues of the glochidia (Figure 2). At that point glochidia began to characteristically ‘wink’ with rapid contractions of their valves, indicating maturity (see also Walker 1981). The larval thread was extremely sticky, as was the vitelline membrane. <FIG 2 ABOUT HERE> Glochidial shell morphology of W. carteri is illustrated in Figure 3. The valves are operated by a single adductor muscle attached to the medial portion of each valve. The shells of W. carteri were subtriangular and inequilateral in shape with a smooth porous surface which lacked surface spikes. The apex of the ventral edge was off-centre and closest to the 4 posterior region of the glochidial shell. The teeth were more or less interlocking when shells of glochidia were closed. Teeth were slightly curved towards the adductor muscle with a concave protuberance on the base