Integrated taxonomy reveals new threatened freshwater mussels (Bivalvia: Hyriidae: Westralunio) from southwestern Australia
Genetic variationThe best fitting substitution models for COI codons 1–3 were identified as TN + F + G4, F81 + F + I, and TN + F, respectively. The maximum likelihood (ML) and Bayesian inference (BI) trees showed similar topologies of the main nodes, although the BI tree displayed greater resolution of the ingroup branches (Fig. 1). Furthermore, the BI tree revealed three monophyletic clades, while two of those clades were merged in the ML tree. Two of the three molecular species delimitation methods (ASAP and TCS) recovered three groups in the BI tree as distinct taxa (Fig. 1), corresponding to the three previously described ESUs27,28. The third method (bPTP) recovered between 8 and 43 groups (mean = 28.03) suggesting that there is evidence of additional genetic differentiation within the three groups identified by ASAP and TCS. The outputs of the three methods are provided in the Supplementary information. The molecular diagnosis uncovered several fixed nucleotide differences COI characters for each taxon (Table 1: “W. carteri” I = 10; “W. carteri” II = 3; “W. carteri” III = 5). There were also 13 fixed nucleotide differences in W. carteri for the 16S gene. The remaining two taxa had no fixed nucleotide differences for the 16S gene.Figure 1Phylogenetic trees obtained by maximum likelihood (left) and Bayesian inference (right) analysis of “Westralunio carteri” mtDNA COI sequences, including support values for the major genetic clades [ultrafast bootstrap values (left) and Bayesian posterior probabilities (right)]. Colour coded bars show support for the three major clades by the species delimitation methods (ASAP = dark shade; TCS = lighter shade). Green = WcI = “W. carteri” I; blue = WcIII = “W. carteri” III; red = WcII = “W. carteri” II. Results of bPTP analysis not shown (see supplementary data). Haplotype names correspond to Benson et al.28. Outgroup taxa are Velesunio ambiguus (Philippi, 1847) (Hyriidae: Velesunioninae) and Cucumerunio novaehollandiae (Gray, 1834) (Hyriidae: Hyriinae: Hyridellini).Full size imageTable 1 Molecular diagnoses of “Westralunio carteri” Evolutionarily Significant Units (ESUs) from southwestern Australia (after Bolotov et al.122 with reanalysis of data from Klunzinger et al.27 and Benson et al.28).Full size tableVariation in shell morphologyBased on results from analyses of variances (ANOVAs), shells of “W. carteri” I were significantly larger (for size metrics total length (TL), maximum height (MH), beak height (BH) and beak length (BL)) and more elongated (i.e., had a lower maximum height index (MHI)) than shells of “W. carteri” II and “W. carteri” II + III combined (Table 2). However, there was no difference in size or shape metrics between “W. carteri” I and “W. carteri” III (Table 2). The lack of significant differences in beak height index (BHI) and beak length index (BLI) among any of the taxa (Table 2) indicates that wing and anterior shell development was not discernibly different between any of the ESUs.Table 2 Shell size metrics [mm], shape indices [%] and scores for the first two principal components (PC) obtained by Principal Component Analysis of shape indices and 18 Fourier coefficients generated by Fourier Shape Analysis for each “Westralunio carteri” species and subspecies-level Evolutionarily Significant Units (ESUs): n, number of specimens measured; minimum (min) to maximum (max) and mean (± standard error (SE)).Full size tableThis pattern was partly confirmed in the principal component analysis (PCA) of these three shell shape indices, where PC1, largely explained by variation in BLI (Fig. 2A), did not differ between the two species (i.e., “W. carteri” I vs. “W. carteri” II + III) or among the three taxa (Table 2). The PC2, largely explained by variation in MHI and BHI (Fig. 2A), differed significantly between “W. carteri” I and “W. carteri” II (Table 2). Accordingly, 70% (70% jack-knifed) of specimens were assigned to the correct species in the corresponding discriminant analysis (DA), whilst this was true for only 55% (54%) at the MOTU-level.Figure 2Scatterplots of the first two PC axes obtained by PCA on (A) calculated shape indices based on shell measurements, and (B) 18 Fourier coefficients for “Westralunio carteri” I, “W. carteri” II and “W. carteri” III. 95% Confidence Intervals are displayed at the species level, i.e., for “W. carteri” I (full line) and “W. carteri” II + III (dashed line). Extreme shell outlines in (B) are depicted to visualise trends in sagittal shell shape, along PC axes.Full size imageThe difference in shell elongation between “W. carteri” I and “W. carteri” II was confirmed by Fourier shape analysis. As visualised by synthetic outlines in Fig. 2B, shell elongation is expressed along the PC1 (explaining 15% of total variation in Fourier coefficients). The PC1 as well as PC2 scores differed significantly between the two species (i.e., “W. carteri” I vs. “W. carteri” II + III) as well as between “W. carteri” I and “W. carteri” II, respectively (Table 2). Combined with synthetic outlines, this indicated a tendency towards a more elongated, somewhat wedge-shaped shell in “W. carteri” I, whilst “W. carteri” II shells tended to be relatively high with a stout anterior margin (Fig. 2B). An analysis of similarities (ANOSIM) analysis on all Fourier coefficients revealed no significant difference between the two species (i.e., “W. carteri” I vs. “W. carteri” II + III; ANOSIM: R = − 0.018, p = 0.097), but did indicate a significant difference between the three ESUs (ANOSIM: R = 0.0625, p = 0.0051). Specifically, “W. carteri” I differed significantly from “W. carteri” II (Bonferroni-corrected p = 0.0009). Only 66% and 65% (62% and 62% jack-knifed) of specimens were assigned to the correct species and taxon in DAs on that dataset, respectively.Taxonomic accountsClass: Bivalvia Linnaeus, 175831.Subclass: Autobranchia Grobben, 189432.Infraclass: Heteroconchia Gray, 185433.Cohort: Palaeoheterodonta Newell, 196534.Order: Unionida Gray, 185433 in Bouchet & Rocroi, 201035.Superfamily: Unionoidea Rafinesque, 182036.Family: Hyriidae Parodiz & Bonetto 196337.Genus: Westralunio Iredale, 19349.Type species: Westralunio ambiguus carteri Iredale, 19349 (by original designation).Redescription: Westralunio carteri (Iredale, 1934)SynonymyUnio australis Lamarck38: Menke39, p. 38, specimen 219. (Non Unio australis Lamarck, 181938).Unio moretonicus Reeve40: Smith41, p. 3, pl. iv, Fig. 2. (misidentified reference to Unio moretonicus Reeve, 186540).Hyridella australis (Lam.38): Cotton & Gabriel42 (in part), p. 156. (misidentified reference to Unio australis Lamarck, 181938).Hyridella ambigua (Philippi26): Cotton & Gabriel42 (in part), p. 157. (misidentified reference to Unio ambiguus Philippi, 184726).Westralunio ambiguus carteri: Iredale, 19349, p. 62.Westralunio ambiguus (Philippi26): Iredale9, p. 62, pl. iii, Fig. 8, pl. iv, Fig. 8. (Non Unio ambiguus Phil. 184726), Iredale43, p. 190.Centralhyria angasi subjecta Iredale, 19349, p. 67 (in part), Iredale43, p. 190.Westralunio carteri Iredale9: McMichael & Hiscock10pl. viii, Figs. 1, 2, 3, 4, 5, 6 and 7, pl. xvii, Figs. 4, 5.Type materialLectotype: AMS C.61724 (Fig. 3A) Westralunio ambiguus carteri Iredale, 19349.Figure 3(A) Westralunio ambiguus carteri Iredale, 1934, Lectotype: Victoria Reservoir, Darling Range, 12 mi E of Perth, AMS C.061724. Detail of fusion in anterior muscle scars from either valve represented by dashed lines and black polygons. Bottom image showing detail of hinge teeth. Photos provided with permission by Dr Mandy Reid, AMS. (B) Valves and detail of sculptured umbo of a juvenile W. carteri from Yule Brook, Western Australia, UMZC 2013.2.9. Photo by Dr Michael W. Klunzinger. (C) Glochidia of W. carteri from Canning River, Western Australia. Photo by Dr Michael W. Klunzinger.Full size imageParalectotypes: AMS C.170635 Westralunio ambiguus carteri Iredale, 19349 (n = 4).Type locality: Victoria Reservoir, Darling Range, 12 miles east of Perth, Western Australia (Fig. 4A).Figure 4(A) Victoria Reservoir, Canning River, near Perth, Western Australia, type locality for W. carteri. Photo by Corey Whisson. (B) Goodga River, Western Australia, type locality for W. inbisi inbisi, at vertical slot fishway where holotype of W. inbisi inbisi was collected from. Photo provided with permission by Dr Stephen J. Beatty. (C) Margaret River, Western Australia, type locality for W. inbisi meridiemus, at Canebreak Pool. Photo by Dr Michael W. Klunzinger.Full size imageLectotype: BMNH 1840–10-21–29 Centralhyria angasi subjecta Iredale (selected by McMichael & Hiscock10).Type locality: Avon River, Western Australia.Material examined for redescription: For W. carteri (= “W. carteri” I), molecular data examined included 52 and 61 individual COI mtDNA and 16S rDNA sequences, respectively, for species delimitation. Additionally, Fourier shell shape outline analysis and traditional shell morphometric measurements were examined from 238 and 290 individuals, respectively. Complete details on all specimens examined are provided in Supplementary Table S1.ZooBank registration: urn:lsid:zoobank.org:act:6B740F4D-40C3-4D6A-8938-B0FD7FD1F6D7.Etymology: The species name carteri is most likely named after the surname of the collector who provided original type specimens to the Australian Museum, although Iredale9 did not specify this as the case. We have applied ICZN Articles 46.1 and 47.144, designating W. carteri as the nominotypical species.Revised diagnosis: Specimens of W. carteri are distinguished from other Australian Westralunio taxa by having shell series that are significantly larger and more elongated than Westralunio inbisi inbisi subsp. nov., but not different from Westralunio inbisi meridiemus subsp. nov. The species has 10 diagnostic nucleotides at COI (57 G, 117 T, 210 G, 249 T, 255 C, 345 G, 423 T, 447 T, 465 A, 499 T) and 13 at 16S (137 T, 155 C, 228 C, 229 T, 260 G, 290 A, 305 G, 307 T, 310 A, 311 C, 321 T, 330 A, 460 A), which differentiate it from its sister taxa, W. inbisi inbisi and W. inbisi meridiemus (each described below) using ASAP and TCS species delimitation models.RedescriptionThis species is of the ESU “W. carteri” I27,28.Shell morphology: Shells of relatively small to medium size, generally less than 70 mm in length, but to a maximum length of approximately 100 mm10,45, MHI 46–89%; anterior portion of shell with moderate development, BLI 22–49%; larger shells with abraded umbos scarcely winged; wing development variable, generally decreasing with size, BHI 76–104% (Table 2). Shell outline oblong-ovate to rounded; posterior end obliquely to squarely truncate, anterior end round; ventral edge slightly curved, nearly straight in larger specimens; hinge line curved, hinge strong. Umbos usually abraded in specimens > 20 mm in length; unabraded umbos with distinctive v- or w-shaped plicated sculpturing (Fig. 3B and Zieritz et al.46). Shell substance typically thick; shells of medium width with pronounced posterior ridge; periostracum smooth, dark brown to reddish, with fine growth lines. Pallial line less developed in smaller specimens and prominent only in large specimens (e.g., > 60 mm TL). Lateral teeth longer and blade-like, slightly serrated to smooth and singular in left valve, fitting into deep groove in right valve; pseudocardinal tooth in right valve coarsely serrated, thick, and erect, fitting into deeply grooved socket in left valve. Anterior muscle scars well impressed and anchored deeply in larger specimens; anterior retractor pedis and protractor pedis scars both small and fused with adductor muscle scar; posterior muscle scars lightly impressed; dorsal muscle scars usually with two or three deep pits anchored to internal umbo region.Anatomy: Supra-anal opening absent, siphons of moderate size, not prominent but protrude beyond shell margin in actively filtering live specimens, pigmented dark brown with mottled lighter brown to orange splotches; inhalant siphon aperture about 1.5 times size of exhalant and bearing 2–4 rows of internal papillae (Fig. 5A); ctenidial diaphragm relatively long and perforated. Outer lamellae of outer ctenidia completely fused to mantle, inner lamellae of inner ctenidia fused to visceral mass then united to form diaphragm; palps relatively small, usually semilunar in shape; marsupium well developed as a distinctive swollen interlamellar space in the middle third of the inner ctenidium of females. Outer ctenidia in both sexes thin, with numerous, short intrafilamentary junctions and few, irregular interlamellar junctions; in females similar, but marsupium has numerous, tightly packed, well-developed interlamellar junctions. Thus, brooding in females is endobranchous.Figure 5Live specimens of actively filtering freshwater mussels in the burrowed position. (A) Westralunio carteri (Iredale, 1934), Canning River at Kelmscott, Western Australia, inhalant siphon with 2–4 rows of papillae oriented toward substrate. Photo by Dr Michael W. Klunzinger. (B) Westralunio inbisi meridiemus subsp. nov., Canebreak Pool, Margaret River, Western Australia; inhalant siphon edges lined with protruding papillae facing towards water surface, away from substrate. Photo by Dr Michael W. Klunzinger.Full size imageLife history: Sexes are separate in W. carteri, and hermaphroditism appears to be rare47,48,49. Males and females both produce gametes year-round but brooding of glochidia appears to be seasonal and ‘tachyticitc’ (i.e., as defined by Bauer & Wächtler19, fertilisation and embryonic development occurring in late winter/early spring and glochidia release in early summer)50. Glochidia are released within vitelline membranes, embedded in mucus which extrude from exhalant siphons of females (i.e., ‘amorphous mucus conglutinates’) during spring/summer. Glochidia attach to host fishes and live parasitically on fins, gills or body surfaces for 3–4 weeks while undergoing metamorphosis to the juvenile stage. Host fishes which have been shown to support glochidia metamorphosis to the juvenile stage in the laboratory include Afurcagobius suppositus (Sauvage, 188051), Gambusia holbrooki (Girard, 185952), Nannoperca vitttata (Castelnau, 187353), Pseudogobius olorum (Sauvage, 188051) and Tandanus bostocki Whitley, 194454 but not Carassisus auratus Linnaeus, 175831 or Geophagus brasiliensis (Quoy & Gaimard, 1824 More
