MALACOLOGIA, 2009, 51(1): 131−141

Redescription of gibbus Spengler, 1793, a west palaearctic freshwater mussel with hookless glochidia

Rafael Araujo*, Carlos Toledo & Annie Machordom

Museo Nacional de Ciencias Naturales (CSIC), José Gutiérrez Abascal 2, 28006 Madrid, Spain

ABSTRACT

As part on an ongoing effort to unravel the and phylogeny of Palaearctic freshwater mussels, this paper describes an overlooked Unio species that inhabits the Atlantic rivers of South Iberia and Morocco, based on morphological, reproductive, and molecular characters. Live specimens of Unio gibbus were recently found in the Barbate River in southwest Spain and in the Beth River in the Sebou basin, northwest Morocco. Phylogenetic relationships inferred from partial COI and 16S rRNA gene sequences point to the recognition of U. gibbus as an evolutionary unit. Genetic divergences with respect to other Unio species ranged from 10.24% and 6.18% (compared to U. tumidus) to 12.91% and 8.89% (compared to U. tumidiformis) for COI and 16S rRNA respectively. In the Spanish specimens, the entire internal cavity of the external demibranchs (homoge- neity) acts as a marsupium (ectobranchy), yet in the Moroccan population, the four gills are occasionally filled with glochidia (occasional tetrageny). Glochidia are rounded triangular, intermediate between the shapes of those in other species of Unio and Potomida. The border of the larval shell bears numerous small lumps, but these fail to form the ventral styliform hook typical of the genus Unio. These two characters, tetrageny and hookless glochidia, both of major significance in phylogenetic studies of the group, are reported here for the first time in the genus Unio. The Spanish populations of U. gibbus should be a main priority for European invertebrate conservation measures. Key words: Unio, hookless glochidia, Iberian Peninsula, Morocco, mitochondrial DNA.

Introduction glochidium, come to light. Recent studies on European unionoid taxonomy necessitate the Most specialists would agree that the seminal formulation of new hypotheses regarding the work of Haas (1969) on unionoid taxonomy number of evolutionary independent taxa in forms the basis for any modern study on the this imperilled mollusc group. With Haas’ work taxonomy and phylogeny of this mollusc group. as the base, recent conchological, anatomical, The genus Unio was considered by Haas reproductive and molecular data have revealed (1969) as a series of 12 “fundamental Unio several new features of the taxonomy of West species” comprised of different “races” or incipi- Palaearctic naiads: (1) the two Iberian Mediter- ent species. Although this author’s work was ranean Unio discussed by Haas (1969) – U. mostly based on conchology, Haas laid down elongatulus penchinatianus Bourguignat, 1865, the basic framework for current freshwater and U. elongatulus valentinus Rossmässler, mussel systematics. In a recent review of the 1854 – are actually synonyms of U. mancus systematics and global diversity of the freshwa- Lamarck, 1819 (Araujo et al., 2005); (2) the ter mussel species, Graf & Cummings (2007) two Iberian Potomida discussed by Haas only considered seven species under the genus (1969) – P. littoralis littoralis Cuvier, 1798, and Unio. Thus, the taxonomy of this polymorphic P. littoralis umbonata Rossmaessler, 1844 – are group now needs to be reconstructed as new synonyms of P. littoralis Cuvier, 1798 (Reis et molecular data and further biological char- al., in review); (3) Unio tumidiformis Castro, acters, such as shell morphology, anatomy, 1885, an overlooked Iberian endemic, is being

*Corresponding author: [email protected]

131 132 Araujo ET AL. redescribed (Reis & Araujo, in review); and (4) ethanol. Total DNA was extracted using the Margaritifera marocana Pallary, 1918, has also ChargeSwitch gDNA Micro Tissue (Invitrogen) been redescribed as a valid species that differs extraction kit or classic phenol/chloroform pro- from M. auricularia (Spengler, 1793) (Araujo et cedures (Sambrook et al., 1989). Partial COI al., in press). and 16S rRNA gene sequences were amplified As part of an ongoing study of the phylogeny by polymerase chain reaction (PCR) using the of the Palaearctic unionoids, the present paper same primers as in Machordom et al. (2003). describes another overlooked Unio species The PCR mix contained 3µl DNA, 5µl of the based on the character set mentioned above. corresponding buffer (with 10x 2mM MgCl2), The species was previously considered a 1µl of dNTPs mix (10mM), 0.8µl of both prim- synonymy of the southwest Atlantic Iberian ers (10µM), 0.3µl Taq DNA polymerase (5U/µl) U. pictorum delphinus Spengler, one of the (Biotools) and ddH2O in a total volume of 50µl. 13 taxa into which Haas (1969) separated The amplification conditions for both genes the west Palaearctic species of Unio pictorum were as follows: 94°C (4 min), 40 cycles of (Linnaeus, 1758). 94°C (45 s), 45°C (1 min), 72°C (1 min), and a final extension at 72°C (10 min). The products were visualised with blue light Materials and Methods in 0.8% agarose gels stained with SYBR Safe (Invitrogen). A co-migrating 100 pb or 1Kb lad- To determine the historical distribution of the der molecular-weight marker served to confirm species, we reviewed Spengler (1793), Bour- correct amplification. The amplified fragments guignat (1864, 1865), Drouet (1893), Simpson (around 700 bp) were purified by ethanol (1900), Haas (1913, 1917, 1969), Pallary precipitation prior to sequencing both strands (1923, 1927, 1936), Azpeitia (1933), Daget using BigDye Terminator (Applied Biosystems, (1998), Soriano et al. (2001), and Knudsen et ABI). Products were electrophoresed on an ABI al. (2003). Recent specimens were obtained by 3730 genetic analyzer (Applied Biosystems). field sampling the Iberian Peninsula and Mo- The forward and reverse DNA sequences rocco in 2006 and 2007; additional material was obtained for each specimen were aligned and kindly provided by collaborators. Specimens checked using the Sequencher program (Gene were collected by wading the rivers, sometimes Code Corporation) after removing primer re- using a water-scoop, or by snorkelling. Some gions. When necessary, Clustal X (Thompson specimens (six from Spain and eight from et al., 1997) was used to align the obtained Morocco) were transported alive in the cold sequences of 16S, but the final alignment was (without water) to the laboratory where they adjusted by eye. were dissected and photographed. For the phylogenetic analyses, we selected Shell characters were examined in adult and GenBank Unio sequences representing the juvenile specimens from the two rivers where type localities of various species (or at least the species were found. Detailed descriptions populations close to them), such as Unio pic- were made of shell shape and outline, colour, torum (Linnaeus, 1758); U. tumidus Philipsson, umbonal sculpture, and hinge characteristics. 1788; U. crassus Philipsson, 1788; U. dougla- All relevant features were photographed. siae Gray, in Griffith & Pidgeon, 1833; U. caffer Anatomical studies were conducted on live Krauss, 1848; and U. mancus Lamarck, 1819, and preserved specimens from the two rivers, and those from the Iberian Peninsula studied paying close attention to characters identified by Reis et al. (in review). Three other species by other authors as taxonomically relevant in belonging to the same family, Potomida lit- unionoids (Ortmann, 1911; Haas, 1924; Nagel, toralis (Cuvier, 1798), Amblema plicata (Say, 1999; Graf & Cummings, 2006). 1817) (both from the subfamily Ambleminae) To characterize the larval stage, glochidia and Anodonta cygnea (Linnaeus, 1758) (sub- were collected from live mussels by their family Anodontinae), were also included in the separation from gravid gills, cleaned with analyses. Margaritifera auricularia (Spengler, KOH and gold-coated for scanning electron 1793), representing the family Margaritiferidae, microscopy. was used as an outgroup. For molecular analyses, two foot samples Analyses were performed on both genes to- from 15 specimens (six from Spain and gether and separately for each gene. A partition nine from Morocco) were obtained and pre- homogeneity test (implemented in PAUP* as served both frozen at -75°C and in absolute ILD test: Mickevich & Farris, 1981; Farris et al., Redescription of Unio gibbus 133

FIG. 1. Map showing the distribution of Unio gibbus. Black points indicate sites where live specimens have been recently discovered - 1: Barbate River; 2: Beth River; 3: Tiflet River Unio[ tifleticusP allary, 1923]; 4: Sous River [Unio (Limniun) foucauldiana Pallary, 1936].

1994) was used to assess congruence among option, for each gene separately and consider- data for the two genes. Phylogenetic analyses ing each codon position in the case of the COI (maximum parsimony – MP, maximum likelihood gene. The Metropolis-coupled Markov chain – ML, and neighbour-joining – NJ) were con- Monte Carlo (MCMCMC) analysis was run for ducted using PAUP* 4.0b10 (Swofford, 2001). 5,000,000 generations, sampling each 100 Equal-weighted parsimony analyses were generations. Burn-in was 10%, after which performed through heuristic searches with TBR likelihood values were stable. Majority rule con- branch swapping and ten random additions. sensus trees were constructed using PAUP. The appropriate model and starting param- eters for ML and NJ analyses were chosen for each of the datasets using the Akaike Infor- RESULTS mation Criterion implemented in Model Test 3.06 (Posada & Crandall, 1998). Maximum Unio gibbus Spengler, 1793 likelihood analysis was performed by heuristic Unio gibbus Spengler, 1793: 64. searches, and the robustness of the topologies Unio turdetanus Drouet, 1893: 66, 67; pl. 1, was assessed by bootstrap replications (1,000) fig. 4. (Felsenstein, 1985). Unio tifleticusP allary, 1923:�������������������� 78. Figured in Pal- Bayesian analyses using MrBayes 3.1.1 lary, 1927: pl. 7, figs. 1, 2. (Huelsenbeck, 2000; Huelsenbeck & Ronquist, Unio (Limniun) foucauldiana Pallary, 1936: 63, 2001) were conducted for each dataset, each 64; pl. 4, fig. 2. gene separately and both together. The Bayes- ian analysis using one cold and three incre- Holotype: Zoological Museum of the Univer- mentally heated chains started from a random sity of Copenhagen, Denmark. ZMUC BIV-434. tree. The models of nucleotide evolution were Illustrated by Haas (1913) and Knudsen et estimated following a model with rate classes, al. (2003). Type locality: “Tranquebar”, India, unequal base frequencies and the “invgamma” probably in error. 134 Araujo ET AL.

Distribution (Fig. 1): Atlantic rivers of south External Morphology (Figs. 2, 3): Shell swol- Iberia and Morocco. Live specimens of U. len, oval or rounded, equivalve, inequilateral, gibbus have been recently found in two rivers usually elevated at posterior end. Outline, es- only: Barbate River, Cádiz, southwest Spain, pecially in juvenile shells, resembling Potomida and two localities in the Beth River, Sebou littoralis. Anterior end rounded in a circular arc, basin, northwest Morocco. In the Spanish river, posterior end rounded but dorsally elevated, the species shares its habitat with Potomida forming a marked keel in ligamental area. Shell littoralis, Unio cf. pictorum and Anodonta cf. thin, light; periostracum flaking in posterior and anatina (Linnaeus, 1758), whereas in Morocco ventral areas. Colour green (more common in it was found living with another species of Unio Moroccan specimens) to brown, sometimes and with Potomida sp. with radial bands of clearer colour. Umbones Pallary (1923, 1927, 1936) cited Unio tifleti- rounded, prominent, never eroded, clearer in cus in the Tiflet River, Sebou basin, and Unio colour that rest of shell. It is common to see the (Limniun) foucauldiana in the Sous River, south glochidium as a protoconch in the apex of the of Agadir, Morocco. umbo (Fig. 3B). Umbo sculpture more marked

FIG. 2. Unio gibbus specimens from different localities. A: Barbate River, Spain; B: Beth River, Morocco; C: Juvenile specimens (Barbate River); D: Umbonal sculpture (Barbate River). Redescription of Unio gibbus 135

FIG. 3. Unio gibbus specimen. A: Detail of the umbonal sculpture; B: Remains of the glochidia present in the umbo. in Spanish specimens, consisting of two or (Fig. 6), intermediate between glochidia shapes three rows of strong tubercula parallel to growth in Unio and Potomida. Border of the larval shell lines of the shell. In juvenile shells, posterior bears numerous small lumps enlarging toward tubercula extend along a diagonal row that is the ventral portion where they become spicules absent in adult shells (Fig. 3A). Left valve with (Fig. 6F, G, H) but never form the ventral styli- two laminar pseudo-cardinal teeth, sometimes form hook typical of the genus. Mean glochidial crenulated, which may or may not be well dimensions (measured by SEM): length: 209.17 separated; posterior tooth beaked and more µm (sd = 2.83; n = 19), height: 211 µm (sd = 3.93; elevated. Two laminar posterior lateral teeth n = 21), width: 67.67 µm (sd = 5.99; n = 3). variable in length, the lower tooth always more The phylogenetic relationships inferred elevated. Right valve with pseudo-cardinal from our data (GenBank accession numbers: tooth generally beaked, sometimes flat, and EU735753 to EU735781) point to the indepen- very tall laminar lateral posterior tooth. Maxi- dence of U. gibbus as an evolutionary unit (Fig. mum measured shell length 85.6 mm. 7). In molecular terms, our results indicate a clear distinction between U. gibbus and the rest Anatomy (Fig. 4): Soft parts typical of Unio. of the species analysed (Fig. 7). The Spanish Papillae of the inhalant siphon generally not specimens showed an absolute lack of genetic bifurcated; exhalant opening confined by two variability, with only one haplotype observed lateral muscular strips with external black for each gene studied. The Iberian and Moroc- trimming, and separated dorsally from the can specimens of U. gibbus displayed a small supra-anal aperture by a bridge shorter than amount of intraspecific divergence (0.37% for the supra-anal aperture. COI), and all shared the same haplotype for the 16S gene. In contrast, divergences were Life Cycle: In the Barbate River, Spain, 30 times higher when compared to other Unio females carried embryos in their gills at the species – 10.24% with respect to the clos- end of February 2006, whereas in the Beth est U. tumidus to 12.91% with respect to U. River, Morocco, ripe glochidia were found in tumidiformis da Silva e Castro, 1885, for COI, specimens collected in early June 2007. In the and 6.18% to 8.89% for 16S with respect to Spanish specimens, the entire internal cavity of the same species, respectively. These results the external demibranchs (homogeneity) acted are based on the analysis of 657 COI and 500 as a marsupium (ectobranchy) (Fig. 5A), but in 16S characters. Although support was low, the Moroccan population the four gills were oc- especially for 16S, global phylogenetic analysis casionally filled with glochidia (occasional tetrag- revealed U. tumidus as the sister species of U. eny) (Fig. 5B, C). Glochidia rounded triangular gibbus (Fig. 7). Moreover, U. gibbus always 136 Araujo ET AL.

FIG. 4. Anatomy of Unio gibbus from the Barbate FIG. 5. Unio gibbus specimens. A: Gravid external River. A: General view; B: Inhalant siphon; C: gill with embryos (Barbate River); B, C: Gravid gills Supra anal aperture. with glochidia (Beth River). appeared as an independent lineage from a douglasiae occupied a basal position in the well-supported cluster that included the Palae- 16S analyses (Fig. 7A). arctic species U. cf. pictorum, U. pictorum, U. Anodonta cygnea appeared as sister group mancus, U. tumidiformis, and U. crassus (Fig. of the genus Unio in both, COI and global 7), and even the African U. caffer according analyses, but surprisingly it was included in the to the COI gene (Fig. 7B). The Chinese U. Unio cluster in the 16S dataset. Redescription of Unio gibbus 137

FIG. 6. Glochidium of Unio gibbus from the Beth River. A, B: General view; C: External view; D: Lateral view; E: Posterior view; F-H: Spicules; I: Ligament.

DISCUSSION that the Tranquebar locality of the Spengler specimen was erroneous, and proposed that it Simpson (1900: 861) considered U. gibbus had actually come from Spain. This is a similar as an indeterminate Oriental . The error to that concerning the locality of the Euro- synonymization of Unio turdetanus Drouet, pean Unio auricularius Spengler (= Margaritifera 1893, with Unio gibbus Spengler, 1793, was auricularia), which obviously did not come from mentioned by Haas (1913), who also suggested East India, as indicated by Spengler (1793). 138 Araujo ET AL.

FIG. 7. Majority-rule consensus trees from Bayesian phylogenetic relationships among members of the Palaearctic genus Unio based on partial sequences of – A: 16S; B: COI; C: 16S + COI. Numbers above branches indicate Bayesian posterior probabilities and Maximum Parsimony bootstrap values; those be- low branches indicate bootstrap values for Maximum Likelihood and Neighbour-Joining treatments. Redescription of Unio gibbus 139

Later, Haas (1917, 1969) listed U. turdetanus the Iberian sample could reflect this reduced Drouet, 1893, and U. gibbus, as synonyms of distribution range, indicating poor conditions in the southwest Atlantic Iberian Unio pictorum the Iberian population resulting in reduction in delphinus Spengler, 1793, one of the 13 taxa the effective population size and a lack of gene into which this author divided U. pictorum. This flow or extinction across some of its potential was probably also why Azpeitia (1933) ascribed distribution. U. gibbus to the same group as U. delphinus It is possible that due to the long ancient Spengler and U. hispanus Rossmässler, 1844, history of the group, true relationships among both synonyms of Unio pictorum, although he its members or their actual biogeographical distinguished U. gibbus from the other spe- patterns are difficult or impossible to recover. cies. Despite this, the results of the few recent stud- Unio (Limniun) foucauldiana, which we syno- ies performed on West Palaearctic unionoids nymize here with U. gibbus, was included by (Reis et al., in review; Araujo et al., in press) Daget (1998) as a synonym of the North African are beginning to unveil a much richer diversity U. elongatulus durieui Deshayes, 1847, one than currently recognized. Thus, further insight of the 17 taxa into which Haas (1969) divided into the evolutionary history of Unio will have to the Mediterranean species U. elongatulus C. wait until areas of such potentially high diversity Pfeiffer, 1825; nevertheless, U. (Limniun) areas as Turkey, Greece, and the Maghreb foucauldiana was not included in Haas (1969). region are subjected to detailed investigation. Surprisingly, Unio tifleticus, the other Pallary Unio gibbus has not been found in recent species that we consider to be a synonym of U. surveys of many Spanish South Atlantic and gibbus, was synonymized by both Haas (1969) Mediterranean rivers, including several tributar- and Daget (1998) with Potomida littoralis fell- ies of the Guadalquivir basin, and the locality mani (Deshayes, 1848), the North African race described by Drouet (1893) for U. turdetanus of P. littoralis. (= U. gibbus). The living population examined According to the characters we examined, es- here was found in a short stretch of the Barbate sentially morphological, molecular, and the glo- River upstream from a large reservoir. The lack chidia, U. gibbus differs considerably from both of genetic variability among these specimens, U. pictorum and U. elongatulus, and logically along with seasonal droughts and excessive from P. littoralis; it is clearly a different species, water extractions from this river, make the probably restricted to some Atlantic Iberian and Spanish populations of U. gibbus a main prior- Moroccan rivers. Our molecular data also sug- ity for invertebrate conservation measures. In gest that U. gibbus is more closely related to U. Morocco, at least in the Beth River, U. gibbus tumidus than to other Unio species. is a very common species. The genetic divergences we found were simi- Besides the external appearance of the shell lar or even greater to those observed among and the molecular markers rendering the spe- other unionoid species (Lydeard et al., 1996; cies unmistakable, the most important charac- Araujo et al., in press), but despite this clear ters that separate U. gibbus from other Unio distinction, the biogeographic implications to species are tetrageny and hookless glochidia, be inferred from the phylogenetic results are both features informative in phylogenetic studies not so obvious. The current distribution of U. of the group. Interestingly, the trait of tetrageny gibbus suggests differentiation in the southern was only detected in one Moroccan specimen, Iberian Peninsula and Morocco most likely, the four gills of which were filled with glochidia, when both areas comprised a single landmass, the external gills being much more swollen than in the Betic-Rifian massif (Steininger & Rögl, the internal ones. Two other gravid Moroccan 1984). Now, given that the Betic-Rifian massif specimens were carrying embryos but only in extended as far as the current Algerian Kabilies the external gills, as also occurred in the three Mountains, the possible spread of U. gibbus gravid Spanish specimens. These observa- to the Mediterranean rivers of North Africa tions suggest that the internal gills may be filled should be considered. This possibility is also later, once the external ones are full: could the suggested by the presence of Unio (Limniun) absence of embryos in the inner gills thus only seurati Pallary, 1936, in Chèlif, Algeria, a very be a question of time, or was the tetragenous similar species to U. gibbus that could probably specimen identified just an isolated case of “oc����- be synonymized with it. casional tetrageny” with no phylogenetic implica- Today’s distribution of U. gibbus is greater in tions? This question will likely be answered as the Maghreb region than on the Iberian Penin- we obtain more information on the reproductive sula. The lower molecular variation noted within strategy of other populations of this species. 140 Araujo ET AL.

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