Expansion and systematics redefinition of the most threatened freshwater mussel family, the Margaritiferidae
Graphical abstract
Introduction
The Unionida is the only strictly freshwater order of bivalves (Bogan, 2008). It is an old and widespread order with approximately 800 described species in 180 genera (Bogan, 2008). Six families are currently recognized within Unionida, but only the Unionidae and the Margaritiferidae are widespread in the Northern Hemisphere (Bogan, 2008). While the Unionidae is extremely diverse (>600 species), until the present study, only 12 species in one genus scattered across North America, Europe, North Africa and Asia had been recognized within the Margaritiferidae (Bolotov et al., 2016, Araujo et al., 2017). Additionally, both families are declining globally and are highly endangered, especially the Margaritiferidae, where all species assessed with sufficient data present a near threatened or threatened conservation status (IUCN, 2018).
Until the end of the twentieth century, the taxonomy and systematics of Unionida had been based primarily on conchological and anatomical characters (e.g. Haas, 1969a, Parmalee and Bogan, 1998, Watters et al., 2009). Due to the better availability of Unionida specimens from North America and Europe, those from tropical and the Southern Hemisphere regions were relatively poorly studied (Simpson, 1900, Simpson, 1914, Ortmann, 1921, McMichael and Hiscock, 1958).
Early systematists encompassed all genera of freshwater mussels, including Margaritana (=Margaritifera) species, within the family Unionidae (Table 1: Lea, 1836, 1838, 1852, 1870; Simpson, 1900, Simpson, 1914, Frierson, 1927). However, in the beginning of the twentieth century, Ortmann (1910) determined that some anatomical characters of some genera were distinct and of prime systematic value. This author erected a new taxon, first as a sub-family, Margaritaninae within Unionidae, but immediately after as a separate family, the Margaritanidae (=Margaritiferidae Henderson, 1929, (1910)), both with the genus and species Margaritana (=Margaritifera) margaritifera (Linnaeus, 1758) as the type. As defined by Ortmann, 1910, Ortmann, 1911a, Ortmann, 1912, the Margaritanidae presented distinct anatomical features from the other Unionidae species, including the lack of discrete apertures separated by mantle fusions, particular gill and marsupium structure, and glochidial (larval) shape (Table 2). Although at first other malacologists did not recognize Margaritiferidae as a separate family (e.g., Simpson, 1914), soon it was accepted by most researchers (e.g. Henderson, 1929), including in the comprehensive classification of the Unionida published by Haas, 1969a, Haas, 1969b. In this fundamental work, the family Margaritiferidae was recognized with nine taxa (five species and four subspecies) under a single genus, Margaritifera, divided in four subgenera: Margaritifera, Cumberlandia, Margaritanopsis and Pseudunio.
During the same period, alternative classifications were published (Modell, 1942, Modell, 1949, Modell, 1964, Starobogatov, 1970, Starobogatov, 1995, Bogatov et al., 2003) based only on few conchological characters that proposed a much larger number of taxa in the Margaritiferidae (Table 1). These studies were controversial and subsequently ignored by most malacologists (e.g., Boss, 1982, Smith, 2001, Graf and Cummings, 2007). Since the beginning of this century, the family Margaritiferidae has been consistently restricted to around 12 species (Smith, 2001, Huff et al., 2004, Graf and Cummings, 2006). Smith (2001), based on morphological characters only, divided the Margaritiferidae into three genera: Pseudunio, Margaritifera, and Margaritanopsis. Soon after, a molecular phylogenetic analysis was published using both nuclear and mitochondrial markers on seven Margaritiferidae species (Huff et al., 2004). Although these phylogenetic analyses presented three clear clades, these did not agree with the genera previously defined by Smith (2001), causing Huff et al. (2004) to conclude that the generic name Margaritifera should be considered for all species. In subsequent phylogenetic studies, the Margaritiferidae has been presented consistently as monophyletic, with a marked genetic structure and divided into three to four major clades; however, most authors have chosen not to discuss its generic assignment keeping Margaritifera as the single genus (Huff et al., 2004, Graf and Cummings, 2007, Araujo et al., 2017). Nevertheless, many North American researchers continued to recognize Cumberlandia as a valid genus (e.g. Watters et al., 2009, Haag, 2012).
Recently, two comprehensive five loci molecular phylogenies on the Margaritiferidae documented several well-supported divergent clades. Bolotov et al. (2016) recognized only three main clades, assigning them as subgenera (Margaritanopsis, Margaritifera, and Pseudunio) of Margaritifera, resembling the previous classification by Haas (1969a). Shortly afterwards, Araujo et al. (2017) described five major divergent clades within the Margaritiferidae, but kept them under the same genus (Margaritifera).
The family Margaritiferidae has a broad but disjunct distribution range in the Northern Hemisphere (Smith, 2001). It presents an enigmatic biogeographic pattern with species aggregations along the western and eastern continental margins and vast distribution gaps in inland areas (e.g., East Europe, Urals and Siberia), possibly reflecting vicariance events driven by plate tectonics (Taylor, 1988, Smith, 2001, Huff et al., 2004). Recently, Bolotov et al., 2016, Araujo et al., 2017 reviewed available biogeographic schemes explaining the origin and expansion routes of the Margaritiferidae and independently provided new fossil-calibrated evolutionary models. However, the time and place of origin of the entire family remained unclear (Bolotov et al., 2016, Araujo et al., 2017). The phylogenetic models placed the origin of the Margaritiferidae in the mid-Cretaceous (Bolotov et al., 2016) or even in the Late Triassic (Araujo et al., 2017). The strong temporal discordance between these fossil-calibrated phylogenies together with significant topological differences and low support values in several deep nodes suggest that both studies need additional taxon samples. Inclusion of Pseudunio homsensis from the Orontes River in Turkey, that had been missing from the previous phylogenetic studies (Bolotov et al., 2016, Araujo et al., 2017), did not help to obtain a fully resolved evolutionary reconstruction for the family, as it appears to be a close relative of P. auricularius (Vikhrev et al., 2017). Additionally, previous analyses also lacked Margaritiferidae taxa from eastern China (i.e., between the Indo-China Peninsula and the Amur River; Smith, 2001, Bolotov et al., 2015, Bolotov et al., 2016). As has already been noted (Smith, 2001, Bolotov et al., 2015), inclusion of newly discovered species from this vast range disjunction is crucial for developing a comprehensive understanding of the biogeography of the Margaritiferidae. Huang et al. (2017) added molecular sequences of Gibbosula rochechouartii to the data set of Araujo et al. (2017) and calculated an updated fossil-calibrated phylogeny placing the origin of the Margaritiferidae crown group in the Late Cretaceous but were not able to obtain a well-resolved biogeographic reconstruction.
A large number of fossil specimens assigned to the Margaritiferidae has been recovered in Europe, Middle Asia, China, Mongolia, Siberia, Japan, North America, and Africa (e.g., Henderson, 1935, Modell, 1957, Martinson, 1982, Ma, 1996, Fang et al., 2009, Van Damme et al., 2015, Bolotov et al., 2016, Araujo et al., 2017). However, recent phylogenetic models were calculated using a limited set of fossil calibrations because the true phylogenetic affinities of many fossil taxa remain unclear due to high conchological variability (Bolotov et al., 2016, Araujo et al., 2017, Huang et al., 2017). The high taxonomic diversity of fossil margaritiferids disagrees with the limited number of extant taxa and likely reflects a lack of critical revisions in systematic paleontology rather than multiple extinction events (Schneider and Prieto, 2011, Bolotov et al., 2016, Araujo et al., 2017). Slow substitution rates in the Margaritiferidae (Bolotov et al., 2016) allow us to expect rather delayed diversification processes within the family, although the diversification rates in margaritiferids have never been tested to date.
Although recent phylogenetic works have increased our knowledge on the position of many Unionida genera from the less studied African and Asian countries (e.g. Pfeiffer and Graf, 2013, Pfeiffer and Graf, 2015, Lopes-Lima et al., 2017a, Bolotov et al., 2017a, Bolotov et al., 2017b), the most comprehensive revision of the Unionidae classification to date placed 42 genera as incertae sedis (Lopes-Lima et al., 2017a). These included Gibbosula (Simpson 1900), whose type species was first described and illustrated by Wood (1815) as Mya crassa from an unknown locality in China and later classified under Gibbosula (i.e. as Gibbosula crassa) within the Unionidae by Simpson (1900). A few years later, another specimen was found in southern China and described as a new species, i.e. Unio (Quadrula) mansuyi Dautzenberg & Fischer, 1908. Simpson (1914) placed this species under Quadrula and did not associate it with G. crassa. A third specimen was described in 1928 and added to Gibbosula (i.e. Gibbosula confragosa Frierson, 1928) based on conchological similarities with G. crassa. In his comprehensive classification of the Unionida, Haas, 1969a, Haas, 1969b considered that Gibbosula had been superfluously created by Simpson and listed it as a synonym of Lamprotula, inside the Unionidae. Additionally, Haas (1969a) listed Dautzenberg & Fischer's species Unio mansuyi as a synonym of Lamprotula crassa.
Simpson (1914) was the first to notice that G. crassa presented some typical margaritiferid conchological features (i.e. mantle attachment scars), but due to other distinct characters (e.g., heavy shell, well developed teeth and deep umbo cavity) it was retained within the Unionidae. Later, Morrison (1975) also noted that Gibbosula had the same characters now known to characterize the Margaritiferidae. However, this information was overlooked by most malacologists who continued to follow Haas (1969a) and kept G. crassa and G. confragosa under Lamprotula (e.g. Prozorova et al., 2005, Graf and Cummings, 2007). Finally, some authors recently described conchological differences between the two Gibbosula species and Lamprotula, and recognized Gibbosula as a separate genus within Unionidae (He and Zhuang, 2013, Graf and Cummings, 2018). Furthermore, based on conchological similarities, a third species of Gibbosula was recently described, i.e. Gibbosula nanningensis (Qian et al., 2015).
The genus Lamprotula was recently revealed to be polyphyletic and divided into Lamprotula s.s. and Aculamprotula (Zhou et al., 2007, Pfeiffer and Graf, 2013). These authors also noted that all species of Lamprotula should be comprehensively analyzed in order to clarify their status and relationships. For instance, based on molecular analyses, Lamprotula rochechouartii has been moved to Margaritiferidae (Huang et al., 2017). In addition, morphological and molecular characteristics of six specimens of G. crassa collected from Bang River, Cao Bang Province, Vietnam in 2016, suggested that the species did not belong to the Unionidae but to the Margaritiferidae (Bogan and Do, 2016). The reassignment of these two Asian species (i.e. L. rochechouartii and G. crassa) from the Unionidae to the Margaritiferidae raises the question whether there are other overlooked species of Margaritiferidae within this group. To address this issue, the congeneric G. confragosa and L. rochechouartii shell types were here analyzed as well as other types of Lamprotula sp. for potentially misplaced margaritiferids.
Under these considerations, the present study aimed to: (i) perform a detailed morphological characterization of collected G. crassa specimens, and available museum specimens of all Margaritiferidae, Lamprotula and Gibbosula; (ii) sequence and characterize the whole F-type mitogenome of G. crassa; (iii) produce a robust phylogeny of the Margaritiferidae using five (nuclear and mitochondrial) markers and discuss the systematics and taxonomy within the family; (iv) compare anatomical, conchological and ecological characters within and among all retrieved clades; and (v) describe the potential origin and ancient radiations of the Margaritiferidae and detect the most probable ancestral geographic areas on the basis of a new multi-locus fossil-calibrated phylogenetic model, using the most complete sampling of taxa to date and an expanded calibration dataset.
Section snippets
Sampling and museum specimens
Six specimens of G. crassa were collected during a survey in northern Vietnam in the Bang River, Cao Bang Province, Vietnam, in 2016. Specimens were deposited as vouchers at the North Carolina Museum of Natural Sciences, United States of America (NCSM 102193, 102194) and at the Institute of Ecology and Biological Resources, Hanoi, Vietnam (IEBR-FM 01-03). Museum specimens of Gibbosula, Lamprotula and Margaritiferidae, including the type specimens of Unio mansuyi and G. confragosa, were analyzed
Mitogenome characteristics
The length of the newly sequenced female mitogenome haplotype of G. crassa (16,196 bp) is within the typical range of Unionida. It includes the 13 protein-coding genes, the gender-specific ORF described for all Unionida mitogenomes with DUI system, 22 transfer RNA (tRNA) and 2 ribosomal RNA (rRNA) genes (Supplementary Fig. 1).
Phylogenetic analyses
The datasets included combinations of individual alignments (COI: 654 bp, 16S: 471 bp, 18S: 1778 bp, 28S: 309 bp, H3: 327 bp). No indels were observed and no stop codons
Definition of the Margaritiferidae
Since the first definition of the Margaritiferidae by Ortmann, its supposed diagnostic characters have varied considerably (Table 2). Graf and Cummings (2006), based on a molecular (COI + 28S) and morphological phylogeny, revised margaritiferid synapomorphies noting that there was no previous consensus on characters diagnosing the family Margaritiferidae. These authors retained only five morphological synapomorphies, two conchological (characters 12 and 27, Table 2) and three anatomical
Conclusions
The current study supports the increase of extant margaritiferid species to 16 and suggests their division into two subfamilies and four genera. Since a better understanding of phylogenetic diversity is central for determining conservation priorities (Lopes-Lima et al., 2017c, Lopes-Lima et al., 2018), the results reported here may be important in the definition of future management strategies devoted to conservation of margaritiferid species. The inclusion of G. crassa, G. polysticta, G.
Acknowledgments
The authors would like to thank the editor Dr. Perez-Losada and two anonymous reviewers for their valuable suggestions and comments. M. Caballer MNHN e-RECOLNAT (ANR-11-INBS-0004) and Ms. Virginie Heros, Mollusks, Museum national d’Histoire Naturelle, Paris are acknowledged for the photographs of the types of Unio mansuyi, Unio rochechouartii, and Unio affinis used in this paper and their assistance with specimens in their collection. Ms. Krasimira (Kasey) Seizova, Drexel University Co-op
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