Liverworts are speciose, morphologically diverse, and members of an ancient lineage that is now recognized as the sister group to all other land plants. Spermatozoid ultrastructural characters have provided insight on interrelationships among plant groups as well as the puzzling placement of some taxa in molecular-based phylogenies. With completion of a comprehensive molecular phylogeny of liverworts based on nuclear, plastid, and mitochondrial sequences, changes in morphology of spermatozoids may be readily tracked across lineages. The research presented herein was conducted to fill in critical data on spermatogenesis in major clades of liverworts and to evaluate the evolutionary changes in these complex cells throughout the phylum. Ultrastructural studies of the locomotory apparatus in mid-stage spermatids and mature spermatozoids were conducted on Aneura pinguis, Scapania nemorea, Calypogeia mulleriana, Bazzania trilobata, and Porella platyphylla. The locomotory apparatus of the taxa examined exhibits the typical liverwort architecture, with a multilayered structure and two staggered flagella that are attached to the spline by dimorphic basal bodies. The locomotory apparatus of Aneura is unique among liverworts in that the two basal bodies are inserted at nearly the same location near the anterior of the MLS. The spermatozoid of all taxa are streamlined and coiled, and contain a long cylindrical nucleus, two mitochondria and a starch-filled plastid. Spermatozoids of Scapania, Porella and Bazzania coil 1.75 revolutions, while Aneura spermatozoids coil nearly 4 revolutions. The plastid terminates the spermatozoid in all but Aneura, where the plastid and nucleus overlap to the terminus. A data matrix was compiled from published data and the present studies, and a list of 21 characters scored for 11 of the most completely studied taxa. Representative taxa were selected from all major clades within the liverwort phylogeny, as well as one moss, one hornwort, and two tracheophytes as outgroups. Mesquite was used to perform an ancestral state reconstruction using maximum likelihood with Mk1and AsymmMk parameter models. Haplomitriopsida taxa (Haplomitrium and Treubia) shared several characters including more than one plastid, more than two mitochondria, wide spline width, and left lateral curve of the lamellar strip; however, no character states were calculated to have a significant proportional likelihood value at the ancestral node. Marchantiopsida taxa (Blasia, Marchantia, Sphaerocarpos) shared several characters including a three microtubules-wide spline aperture, a notch in the lamellar strip, and a right and left taper in shape to the lamellar strip, all of which were supported with significant proportional likelihood values at the ancestral node. Jungermanniopsida taxa (Pellia, Pallavicinia, Aneura, Porella, Bazzania, Scapania) possessed a right to left taper of the lamellar strip and a spline that attached tangentially to the nucleus and were supported by significant proportional likelihood values at the ancestral node. BayesTraits was used to give estimated values of characters for the liverwort ancestor using both the directional and random walk models. Values included a spline width of 48 microtubules, a lamellar strip length of 0.933 µm, and lamellar strip width of 1.427 µm. In addition to possessing features that have not been documented, Aneura spermatozoids share features with distantly related Haplomitriopsida including the absences of a Fibrillenscheide and the lack of a narrow spline shank.

Ultrastructural observations reveal that the spermatozoid of Lycopodium obscurum is crescent shaped and contains two posteriorly directed flagella that are inserted at the front of the cell. The nucleus is broad and elongated with a narrow posterior projection or nuclear diverticulum. Spline microtubules (MTs) number 180 at their maximum and provide the framework for the cell. These MTs extend from the anterior of the locomotory apparatus and along the outermost surface of the nucleus, with a central shank of 14-17 MTs encircling the cell for at least one-third gyre beyond the nucleus. The two basal bodies are slightly staggered and positioned at the front of the cell over a highly elongated multilayered structure (MLS). The MLS extends laterally around the cell anterior and curves posteriorly over the nucleus. One large anterior mitochondrion is situated subjacent to the MLS, while numerous small mitochondria are scattered near or among the lobes of the single plastid. The plastid rests on the inner nuclear surface and contains numerous large starch grains. This cell differs from that of L. cernuum, the only other species of Lycopodium examined to date, in that it is more elongated and has an anterior-posterior orientation of the nucleus, basal bodies, MLS, and spline. Comparisons with coiled gametes of bryophytes and Selaginella suggest that some degree of coiling and cell streamlining may be ancestral in archegoniate spermatozoids.

At maturity, spermatozoids of the green alga Chara vulgaris are biflagellated, contain little cytoplasm, and coil for approximately 2 1/4 gyres within the mother cell wall. The anterior of the cell contains an ovoid headpiece anchoring two slightly staggered basal bodies that are positioned above and directly in front of approximately 30 linearly arranged mitochondria. An elongated stellate pattern occupies the transition zone between the BBs and axonemes. Flagella emerge from the cell just in front of the nucleus and encircle the full length of the spermatozoid. The spline comprises a maximum of 38 microtubules surrounding the anterior mitochondria and gradually decreases posteriorly to a minimum of 11. The dense nucleus is narrow, cylindrical, and occupies the central revolution of the cell. Six starch-laden plastids and associated mitochondria are linearly arranged at the cell posterior. Phylogenetic analyses of charalean taxa and archegoniates based on spermatogenesis strongly support the order Charales, with Nitella as the sister group to Chara. Diagnostic features of Chara spermatozoids include absence of a lamellar strip and axonemes embedded in the cell for almost the entire length of the anterior mitochondria. Potential relationships among Charales, Coleochaetales and archegoniates are evaluated in regards to the probable course of evolution of streamlined biflagellated gametes.