Description of the glochidia of Westralunio albertisi Clench, 1957 (Bivalvia: Unionida: Hyriidae) from the Fly River system, Papua New Guinea

ABSTRACT Larvae (glochidia) of the freshwater mussel, Westralunio albertisi were obtained from the marsupia of a preserved female at the Western Australian Museum. Glochidial shells of W. albertisi are subtriangular and scalene in shape outline with a convoluted surface dotted with pores and have a protruding ventral apex. Glochidial shells (n = 60) measured 260.0 μm long (L) (±1.11 SE), 233.1 μm high (H) (± 1.32 SE), with a size of 246.6 μm (± 0.99 SE), hinge length (Hg) of 183.6 μm (± 0.99 SE), larval tooth length (LTL) of 53.6 μm (± 0.84 SE) and angle of obliquity (Á) of 12.9° (± 0.28 SE). Larval teeth are singular in each valve, lack microstylets, have convex or concave basal protuberances on opposing valves and terminate as blunt outward facing spoon-like to peg-like cusps. In comparison to Westralunio carteri, W. albertisi is smaller (d.f., 178; P < 0.001) for L (t = 33.85), H (t = 12.80), Size (t = 28.98), Hg (t = 21.32), H/L (t = −15.06) and Hg/L (t = −4.21). Westralunio albertisi also has a more acute Á than W. carteri (d.f., 71; t, 10.17; P < 0.001), but displays no difference in LTL (d.f., 6; t, 0.16, P = 0.44).


Introduction
Present day freshwater mussels (Bivalvia: Palaeoheterodonta: Unionida) inhabit inland waters of every continent apart from Antarctica, with an estimated 958 species (Graf and Cummings 2021).Extant Hyriidae occur in the Southern Hemisphere, found in Australasia (Australia, New Guinea, New Zealand and Solomon Islands) and South America, comprised of 15 genera and 93 species (Marshall et al. 2014;Walker et al. 2014;Graf and Cummings 2021;Klunzinger et al. 2022;Ponder et al. 2022).Like Unionidae and Margaritiferidae, hyriids bear glochidia larvae, many of which are parasitic on fishes, although some South American taxa have foregone parasitism (Walker et al. 2014;Miyahira et al. 2017).Glochidia develop from embryos brooded in the marsupia of female ctenidia and parasitic glochidia are released when they reach a mature stage, ready to attach to a host fish or amphibian (Ponder et al. 2020).Glochidia release mechanisms are variable and sometimes highly specialised (Barnhart et al. 2008).In Australasian Hyriidae, glochidia are released functionally, in mesoconglutinates, which resemble host prey items (Melchior et al. 2021a), simply broadcast into the water column (Melchior et al. 2021a(Melchior et al. , 2021b;;Klunzinger et al., unpublished data) or in amorphous mucus conglutinates, a passive host entanglement strategy (Klunzinger et al. 2012(Klunzinger et al. , 2013;;Klunzinger 2020;Melchior et al. 2021aMelchior et al. , 2021b)).

Materials and methods
Glochidia were dissected from the marsupia of ethanol preserved W. albertisi (Western Australian Museum Registration No. S72148) by means of a pipette, then placed on a glass slide and examined under a light microscope to determine stage of maturation.Because several glochidia appeared to be open and some were floating freely in ethanol within the preservation jar and had what appeared to be fully formed larval teeth, it was assumed that they had been released and were presumably mature (see Klunzinger et al. 2012, 2013and Klunzinger 2020 for description of glochidial maturation).Several subsamples of glochidia were siphoned from the gravid female and specimen jar using a glass pipette and deposited onto specimen stubs with double sided carbon tape and allowed to dry overnight.The following day, specimens were photographed using a Hitachi TM 3030 Plus Scanning Electron Microscope (SEM) and associated software at the Western Australian Museum.Terminology used to describe glochidial morphology follows Jones et al. (1986), Mansur (1999), Pimpão et al. (2012), Klunzinger et al. (2013), Klunzinger (2020) and Melchior et al. (2021a).Glochidia were measured to the nearest 1 µm from scale-imprinted SEM images for length (L), height (H), hinge length (Hg) and larval tooth length (LTL) and, to the nearest 1 degree (°), for angle of obliquity (Á) (see Figure 1).A size metric was determined as the mean of L and H for each individually measured glochidium (Klunzinger 2020;Melchior et al. 2021a).
Data on glochidial shell measurements (L, H, and Hg) of W. carteri (Klunzinger et al. 2013) were used for comparison; 'size' was also determined from the data of Klunzinger et al. (2013) and unpublished SEM images of shell outlines of this species were measured for Á and LTL.Where two opposing larval teeth could be accurately measured from an individual glochidium, the reported LTL value was the mean of the two larval teeth.However, in the case where only one larval tooth could be measured on an individual glochidium it was reported as the LTL value.
Shell measurements of W. albertisi and W. carteri glochidia were compared statistically by independent samples t-test using SPSS 29.0 statistical software.Means with P < 0.05 were considered significantly different.

Results
The glochidial shell morphology of W. albertisi is illustrated in Figure 2. The valves are operated by a single adductor muscle attached to the medial portion of each valve.The shells of W. albertisi glochidia are subtriangular and scalene in shape with a somewhat convoluted to smooth surface dotted with pores which lack surface spikes.The ventral apex of the shell protrudes and is located off-centre, closest to the posterior region of the glochidial shell.Larval teeth are singular in each valve, slightly curved towards the adductor muscle with a concave protuberance on the base of the right valve tooth and a convex protuberance on the base of the left valve, terminating in an outward facing blunt spoon-like to peg-like cusp.A summary of morphometric measurements of glochidia shells of W. albertisi is presented in Table 1 along with its comparative congener W. carteri.Data for individually measured glochidia of both species are available in the supplementary data.Samples of glochidia of W. albertisi examined (n = 60) had a mean L of 260.0 µm (± 1.11 SE) μm, mean H of 233.1 µm (± 1.32 SE) μm, mean 'size' of 246.6 µm (± 0.99 SE), Hg of 183.6 µm (± 0.99 SE), LTL of 53.6 µm (± 0.84 SE) and Á of 12.9°(± 0.28 SE).Westralunio albertisi is significantly smaller than its congener, W. carteri, in L, H, Size, Hg, H/L and Hg/L (Table 1).Westralunio albertisi also has a more acute Á than W. carteri, giving a less scalene sub-triangular shape outline, but there is no difference in LTL between the two species (Table 1).

Discussion
The glochidia of W. albertisi are morphologically distinct from those known from other species of freshwater mussels.In particular, the larval teeth of this species have a unique morphology.The cusps of individual larval teeth curve outwards, unlike the glochidia of other Hyriidae (e.g., Parodiz and Bonetto 1963;Walker 1981;Humphrey 1984;Widarto 1993 (Parodiz and Bonetto 1963;Walker 1981;Humphrey 1984;Widarto 1993;Klunzinger 2020), W. albertisi has larval teeth with blunt, spoon-like cusps which face outward, pointing away from the dorsal hinge.Westralunio carteri differs from other velesunionine glochidia in having straighter, more Table 1.Glochidia dimensions in freshwater mussels (Bivalvia: Hyriidae) from this study.Values presented are means ± standard errors with n representing the number of samples within each taxon measured for each variable.Ranges are provided in parentheses.H, height; L, length; Hg, hinge length; LTL, larval tooth length; Á, angle of obliquity.Taxonomy follows Walker et al. (2014).Shell measurements for W. carteri, except LTL and Á, were also reported by Klunzinger et al. 2013.Data of individually measured glochidia are provided in supplementary material.Results of independent samples t-test are provided with degrees of freedom (d.f.), t-statistic and P-value.Atkins (1979) showed that larval teeth of H. drapeta from southwestern Victoria are s-shaped, like A. jacksoni, A. pertexta pertexta, V. angasi and V. ambiguus, discussed above.Given that glochidia of W. albertisi were obtained from museum preserved material, the natural larval release mechanism could not be observed and the presence or absence of a larval thread in this species could not be confirmed.However, if the species follows a similar strategy to its other velesunionine counterparts, it may be reasonable to assume that the presence of a larval thread and release by amorphous mucus conglutinates (see Walker 1981;Klunzinger et al. 2012Klunzinger et al. , 2013;;Klunzinger 2020) and a passive entanglement infection strategy is likely.Given that A. jacksoni, V. angasi, V. ambiguus and W. carteri are host generalists, using multiple species of host fishes for parasitism and, presumably, dispersal (Walker 1981;Humphrey 1984;Widarto 1993;Klunzinger et al. 2012), it is quite possible that W. albertisi is also a host fish generalist, but this remains unknown.Furthermore, the relatively large size of W. albertisi suggests that it is likely a fin parasite, unlike small glochidia which are usually gill parasites; fin parasites, more often than not, use a passive entanglement mechanism for attachment to their host (Barnhart et al. 2008;Melchior et al. 2021aMelchior et al. , 2021b)).
The position of W. albertisi and W. flyensis within the genus Westralunio is unclear (Walker et al. 2014;Klunzinger et al. 2022).Phylogenetic analyses reported by Graf et al. (2015) and Santos-Neto et al. (2016) showed that W. carteri is separate to other Velesunioninae.Additionally, Zieritz et al. (2013) and Klunzinger et al. (2022) showed that the shell sculpture pattern exhibited by juvenile W. carteri, and indeed glochidia characteristics of W. carteri, might be used to argue that the genus does not belong in the same subfamily as other Velesunioninae.Unpublished genetic data (Jones, Klunzinger, Wilson and Kirkendale, in prep.) also suggest that W. albertisi and W. flyensis may be a separate genus to Australian Westralunio taxa and glochidia data on W. albertisi, presented herein, will likely be useful in arguing for a new taxonomic arrangement.

Figure 1 .
Figure 1.Morphometric measurements of glochidia shells.A, L, length and Hg, hinge length, measured from anterior to posterior; H, height, measured from hinge to ventral apex; B, LTL, larval tooth length, measured from the base of the junction with each valve to the terminal point of each larval tooth (lt); C, Á, angle of obliquity, measured in degrees (°), as the angle between the line that joins the ventral point (a) to the point perpendicular to the hinge (b) and the line that joins the ventral point (a) to the middle of the hinge line (c).

Figure 2 .
Figure 2. Scanning electron microscopy of glochidia of Westralunio albertisi.A, Shell shape subtriangular, scalene with ventral edge apex protruding and located off-centre and closest to the anterior end of a left valve; shell surface convoluted to smooth and dotted with pores; B, Ventral view, valves open, anterior end to right; position of adductor muscle (am) indicated; larval teeth enlarged in C and D, convex basal protuberance (cxp) and concave basal protuberance (cvp) indicated by arrows for each larval tooth; C, Right larval tooth, terminating to a relatively broad, blunt outward facing peg-to spoon-like cusp; D, Left larval tooth, terminating to a relatively broad, blunt spoon-like cusp.