Molecular Systematics of Recent and Pleistocene Brachiopods

Enzyme linked immunosorbent assay (ELISA) of shell intracrystalline proteinaceous macromolecules has been applied to investigate the phylogenetic relationships among 53 living articulate brachiopods (Class Articulata), covering all the living superfamilies and most of the living families. One of the articulate superfamilies, the Cancellothyridoidea, has been investigated by a combined immunological and morphometric approach, with additional materials to include most of the living species of Terebratulina, which is one of the most prolific among the Cenozoic brachiopod genera. The immunological techniques have also been applied to the phylogenetic investigation of Pleistocene brachiopods, including 2 extinct species. Both living and fossil brachiopod shell intracrystalline macromolecules have been analysed using various biochemical techniques. Antisera prepared against 14 living and taxonomically-diverse species allowed ordinal and superfamilial discriminations within the class, and using the more specific pre-absorbed antisera, it was possible to obtain precise species-level information with taxonomic consistency. These experiments revealed that the examined living terebratulide families belonged to one of the following four groups (expressed in provisional superfamilial denotations), which were further clustered into three major groups (provisionally considered as subordinal rank) of a trichotomous relationship: (A) Cancellothyridoidea (short-looped): Cancellothyrididae, Chlidonophoridae; (B1) Terebratuloidea (short-looped): Terebratulidae, Dyscoliidae; (B2). 'Kraussinoidea' (long-looped): Kraussinidae, Megathyrididae, Macandreviidae, Ecnomiosidae; (C) Terebratelloidea (long-looped): Terebratellidae, Laqueidae, Dallinidae. These results were compared and intercalated with both morphological data and the fossil record reaching the following phylogenetic interpretation: The ancestors of the three groups radiated in the early Devonian, and each of those gave rise to the group A, B1, and C in the Mesozoic; in the Triassic another long-looped lineage (possibly the extinct Zeilleriidae) diverged from the short-looped B1 lineage, and this long-looped stock gave rise to the Kingenidae and group B2, probably by processes of neoteny. This scenario suggests that the long loop evolved at least twice independently in the Terebratulida, and also highlights the enigmatic origin of the Cancellothyridoidea. Among the Cancellothyridoidea, 21 living species and subspecies of Terebratulina were assigned into two major phylogenetic groups, which were further divided into 7 subgroups on the basis of the immunological, morphometric, and other data. Degrees of molecular variations between some Terebratulina species were comparable with those between families in other terebratulide superfamilies, suggesting the existence of deep branching events within the living Terebratulina. Fractionations of Terebratulina shell intracrystalline macromolecules by various liquid chromatography techniques revealed several discrete components, including at least one proteinaceous component, most of which were antigenic. Some of these components were revealed to have been preserved more or less intact in a Pleistocene sample, in terms of the molecular weight, amino acid composition, and antigenic properties. The fossil sample also contained degradation and condensation products. Immunological assays on Pleistocene materials from a series of horizons indicated a progressive decay of the macromolecules through time, but the novel serial antisera concentration method demonstrated that even 1 Myr old shells contained lineage-specific molecular information, which allowed family- to species-level phylogenetic reconstructions for the extinct terebratulide species, Kikaithyris hanzawai and an undescribed species of Terebratulina