Sex Plant Reprod (2011) 24:97–112 (Figs. 1, 5, 6a, b, 7; Kirk et al. 1993). The fact that the gonidia develop directly without ﬁrst developing functional ﬂagella is the criterion for ‘‘complete’’ germ–soma division of labor. Mutational analysis of the genetic program for germ– soma differentiation in V. carteri identiﬁed three types of key genes: gonidialess A (glsA), regenerator A (regA), and late gonidia (lag) genes. Based on the mutational and molecular genetic results, a minimal model for the genetic program of germ–soma differentiation was established by David Kirk (Fig. 7; Kirk 1998, 2001, 2005; Kirk and Kirk 2004). In this model, the glsA gene acts during cleavage to permit asymmetric division and production of the large– small sister-cell pairs (Miller and Kirk 1999). In the small (somatic) cells, the regA gene acts as a transcriptional Fig. 7 Minimal model for the genetic program of germ–soma differentiation in V. carteri. Three key gene types are involved in programming differentiation: glsA, lag, and regA. At the 32-cell stage, the glsA gene acts to promote the asymmetric divisions that produce large–small sister-cell pairs. The lag genes then act in the large cells to repress the development of somatic characteristics, while the regA gene acts in the small cells to repress reproductive development. Adapted from D. L. Kirk (Kirk 2001, 2005; Kirk and Kirk 2004) 105 repressor to prevent all aspects of reproductive develop- ment (e.g., by repressing chloroplast biogenesis), while somatic genes are transcribed (Meissner et al. 1999; Kirk et al. 1999). The lag genes act as transcriptional repressors in the large cells (gonidia) to prevent the formation of somatic characteristics such as ﬂagella and eyespots, while gonidial genes are transcribed (Kirk 1998). It is likely that the difference in cell size at the end of cleavage determines whether regA or the lag genes will be activated. However, it remains unclear how cell size is signaled to the regula- tory program, which other components are involved and how the identiﬁed key genes ﬁt into a larger regulatory network. Several paralogous genes that encode proteins resem- bling regA have been identiﬁed both in V. carteri [e.g., the regA-like sequence D (rlsD) gene] and C. reinhardtii [e.g., RLS1] (Kirk et al. 1999; Duncan et al. 2006, 2007). However, Chlamydomonas does not have a gene that is orthologous to regA (Duncan et al. 2007). The closest homolog of Volvox regA in C. reinhardtii is RLS1, but the orthologous gene of C. reinhardtii RLS1 in Volvox is rlsD (Nedelcu 2009). These ﬁndings might be an indication of gene duplications in the common unicellular ancestor of Volvox and Chlamydomonas and then divergence of one of the gene copies, regA. It was speculated that the ancestor of regA might have acted in a stress-activated pathway that led to the repression of growth and cell division in response to energy or nutrient deprivation (Nedelcu 2009; Miller 2010). The paralogous genes of regA both in Volvox and Chlamydomonas might still have this function, while the function of regA might have changed and then was used to co-opt the entire pathway to repress growth and division in a developmental context (Nedelcu 2009; Miller 2010). In contrast to the regA gene, the glsA gene of V. carteri has an orthologous gene in C. reinhardtii, GAR1 (Cheng et al. 2003). GAR1 was shown to function just like glsA: When transformed into Volvox glsA mutants, it rescued the wild- type phenotype (Cheng et al. 2003). In the evolution of the volvocine lineage, glsA obviously was adopted for its novel function in germ–soma differentiation with no signiﬁcant changes. Key evolutionary innovations in the sexual life cycle In the case of C. reinhardtii, the usual trigger for the switch to the sexual mode of reproduction is a lack of nitrogen (Sager and Granick 1954), indicating nutrient deﬁciency; in the case of V. carteri, the transition can be triggered by heat shock (Kirk and Kirk 1986), indicating drying up of the pond. The sexual cycle produces heavily walled, dor- mant zygotes (zygospores) that can resist tough conditions like drought, heat, and cold for a long period of time. 123