Fossil-calibrated molecular clock data enable reconstruction of steps leading to differentiated multicellularity and anisogamy in the Volvocine algae

Abstract

Background: Throughout its nearly four-billion-year history, life has undergone evolutionary transitions in which simpler subunits have become integrated to form a more complex whole. Many of these transitions opened the door to innovations that resulted in increased biodiversity and/or organismal efficiency. The evolution of multicellularity from unicellular forms represents one such transition, one that paved the way for cellular differentiation, including differentiation of male and female gametes. A useful model for studying the evolution of multicellularity and cellular differentiation is the volvocine algae, a clade of freshwater green algae whose members range from unicellular to colonial, from undifferentiated to completely differentiated, and whose gamete types can be isogamous, anisogamous, or oogamous. To better understand how multicellularity, differentiation, and gametes evolved in this group, we used comparative genomics and fossil data to establish a geologically calibrated roadmap of when these innovations occurred.

Results: Our ancestral-state reconstructions, show that multicellularity arose independently twice in the volvocine algae. Our chronograms indicate multicellularity evolved during the Carboniferous-Triassic periods in Goniaceae + Volvocaceae, and possibly as early as the Cretaceous in Tetrabaenaceae. Using divergence time estimates we inferred when, and in what order, specific developmental changes occurred that led to differentiated multicellularity and oogamy. We find that in the volvocine algae the temporal sequence of developmental changes leading to differentiated multicellularity is much as proposed by David Kirk, and that multicellularity is correlated with the acquisition of anisogamy and oogamy. Lastly, morphological, molecular, and divergence time data suggest the possibility of cryptic species in Tetrabaenaceae.

Conclusions: Large molecular datasets and robust phylogenetic methods are bringing the evolutionary history of the volvocine algae more sharply into focus. Mounting evidence suggests that extant species in this group are the result of two independent origins of multicellularity and multiple independent origins of cell differentiation. Also, the origin of the Tetrabaenaceae-Goniaceae-Volvocaceae clade may be much older than previously thought. Finally, the possibility of cryptic species in the Tetrabaenaceae provides an exciting opportunity to study the recent divergence of lineages adapted to live in very different thermal environments.

Keywords: Ancestral state reconstruction; Cellular differentiation; Fossil calibration; Molecular clock; Multicellularity; Phylogeny; Volvocine algae.

Fig. 1.

Cladogram and photographs of volvocine genera. Depicted evolutionary relationships derived from Lindsey et al. [11] with Goniaceae modified to be monophyletic based upon more recent evidence from Ma et al. [12]. The three multicellular volvocine families (Tetrabaenaceae, Goniaceae, and Volvocaceae) are represented by orange, purple, and green font and borders. Unicellularity is denoted by black font and borders. Blue lines correspond to anisogamous lineages, and blue to pink gradient lines represent lineages in which oogamy has evolved. A single asterisk (*) denotes somatic cell differentiation, and double asterisks (**) denote complete germ-soma differentiation. Superscript letters correspond to their respective photographs. Photographs are arranged in the following order: (A) Tetrabaena socialis, (B) Chlamydomonas reinhardtii, (C) Gonium pectorale, (D) Astrephomene gubernaculifera, (E) Platydorina caudata, (F) Colemanosphaera charkowiensis, (G) Pandorina morum, (H) Volvulina compacta, (I) Yamagishiella unicocca, (J) Eudorina elegans, (K) Pleodorina starrii, (L) Volvox carteri. Photos are not to scale. Figure Credit for (B) and (C): Deborah Shelton

Fig. 2.

Time-calibrated phylogeny of the Archaeplastida. Branching order of the tree was inferred under maximum-likelihood analysis from an aligned amino acid, concatenated dataset of 263 nuclear genes. Numbers on branches represent bootstrap and posterior probability values, respectively. Branch lengths, corresponding to time, were inferred under the CIR relaxed clock model using 16 most clock-like genes as determined by Sortadate. Blue bars correspond to the inferred 95% HPD interval for each node. Red bubbles correspond to calibrated nodes (Table 1), and blue bubbles correspond to key divergences among the volvocine algae (Figure S4B). Members of the multicellular volvocine algae (Tetrabaenaceae, Goniaceae, and Volvocaceae) are denoted in orange, purple, and green. Taxa in black font are unicellular volvocine algae

Fig. 3.

Estimated divergence times of the volvocine algae. Branching order of the tree was inferred under maximum-likelihood analysis from an aligned amino acid, concatenated dataset of 263 nuclear genes. Numbers on branches represent bootstrap and posterior probability values, respectively. Branch lengths, corresponding to time, were inferred under the CIR relaxed clock model using 16 most clock-like genes as determined by Sortadate. Blue bars correspond to the inferred 95% HPD interval for each node. Green bubbles correspond to a developmental trait gain, and red bubbles corresponds to loss of a trait. The figure table lists the 12 developmental traits identified by Kirk in their original order. Blue bubbles indicate key divergences in the volvocine algae. Members of the multicellular volvocine algae (Tetrabaenaceae, Goniaceae, and Volvocaceae) are denoted in orange, purple, and green. Taxa in black font are unicellular volvocine algae

Fig. 4.

Sexual trait gain and loss in the volvocine algae. Branching order is of a collapsed tree inferred under maximum-likelihood analysis from an aligned amino acid, concatenated dataset of 263 nuclear genes. Numbers on branches represent bootstrap and posterior probability values, respectively. Branch lengths, corresponding to time, were inferred under the CIR relaxed clock model using 16 most clock-like genes as determined by Sortadate. Blue bars correspond to the inferred 95% HPD interval for each node. Green bubbles correspond to a sexual trait gain, and red bubbles corresponds to loss of a trait. The figure table lists the 7 mapped sexual traits and their acronyms. Pink branches denote lineages where anisogamy evolved. Members of the multicellular volvocine algae (Tetrabaenaceae, Goniaceae, and Volvocaceae) are denoted in orange, purple, and green. Taxa in black font are unicellular volvocine algae