The pollen tube is a unique feature of male gametophytes of seed plants. In cycads and Ginkgo, pollen tubes are long-lived and function solely as haustorial, highly branched structures that grow invasively into female tissues [1–3]. In conifers and Gnetales pollen tubes function in a new way to deliver non-motile sperm to the egg (siphonogamy), while generally retaining a haustorial growth pattern [2, 3]. Flowering plant (angiosperm) pollen tubes have lost most features of haustorial growth - their pollen tubes are typically short-lived and seem to function exclusively to deliver sperm to the egg [4, 5]. The origin of siphonogamy has been held up as a classic example of exaptation , because the plesiomorphic function of the pollen tube - nutritional support for the male gametophyte - was subsequently co-opted for a novel role in sperm delivery . Yet siphonogamy is clearly a complex process, and it is not at all obvious which aspects have common origins, which represent modifications of an ancestral pattern, and which have arisen independently in separate lineages [1, 3, 4, 7]. Understanding the homologies of pollen tube structure and growth pattern may provide deeper insights into the origin(s) of this remarkable innovation.
Angiosperm pollen tubes have a unique wall structure. Their thin growing tip is comprised almost entirely of pectins. Just behind the pectic tip, cellulose synthases operate to form a very thin, pecto-cellulosic primary wall. Then, still in the subapical region, (1,3)-β-glucan (callose) is synthesized beneath the thin primary wall to form a thick layer . The mature pollen tube wall of most angiosperms is primarily made of callose (81% by weight in Nicotiana; ref. 9). As an amorphous polysaccharide, callose can be synthesized more rapidly than an equivalent weight of fibrous cellulosic cell wall  and it provides resistance to tensile and compression stress . Callose also severely reduces wall permeability and since angiosperm pollen tube walls are also prone to forming septae ("callose plugs") , the plesiomorphic haustorial function of tubes is largely precluded. These patterns are general features of all angiosperms, from Amborella and water lilies to Arabidopsis and maize [4, 12]. Yet, despite their ubiquity, the ancestral function of callose walls and plugs is not obvious. Tubes that lack callose in their walls retain their function in some derived eudicot lineages, such as Lamiales  and in an Arabidopsis mutant line [14, 15], though they have reduced competitive ability in the latter .
Pollen tubes in ovules of gymnosperms rarely contain callose in lateral walls, and callose plugs have never been reported . Callose is found in the tip wall of growing pollen tubes of some conifers , a pattern never seen in angiosperms. Studies of in vitro- grown gymnosperm pollen tubes do sometimes find callose (or mixed-glucans) in lateral tube walls [17, 18]. Importantly, the deposition of callose is generally transient in gymnosperm male gametophytes and its extent and location varies even among closely related species [17–20]. Such transient and variable phenotypes contrast with the relatively invariant and persistent expression pattern seen in angiosperm pollen tubes.
Callose synthesis is mediated by the enzyme, callose synthase, encoded by the callose synthase gene, and originally described as a glucan synthase-like gene (GSL) in Nicotiana alata. Hong et al. (2001) named the callose synthase gene family CalS after identifying 12 gene family members in Arabidopsis thaliana. AtCalS5 and its characterized orthologues (NaGSL1 from N. alata) have been directly linked to pollen tube wall formation and callose plug deposition, as well as to pollen exine development [14, 15, 23, 24].
CalS5 appears to have an ancient origin by duplication. A comparative phylogenetic analysis of all CalS paralogs from the genomes of the moss, Physcomitrella patens and Arabidopsis found that AtCalS5 was more closely related to a Physcomitrella CalS gene copy (PpCalS5) than to any other Arabidopsis paralog. Because callose was observed in the moss spore aperture region and PpCalS5 was identified as a putative orthologue to AtCalS5, PpCalS5 was hypothesized to play a role in moss spore germination . If so, then CalS5 involvement in pollen tube growth may ultimately derive from a more ancient function involving the germination process. As such, changes in gene regulation were likely prerequisites for the acquisition of novel callose deposition patterns in angiosperm pollen tube walls. Alternatively, the patterns arose via duplication and functional divergence of a CalS gene within seed plants, or perhaps within the stem lineage leading to angiosperms.
In this paper we present molecular evidence that CalS5 orthologues are expressed in mature pollen and pollen tubes of several extant early-diverging angiosperms in Nymphaeales and Austrobaileyales and likely also in Amborella trichopoda. CalS5 orthologues are also expressed in mature gymnosperm pollen, including one siphonagam (Gnetum) and one non-siphonogam (Ginkgo). In the siphonogamous conifer, Pinus, we report a potentially unique CalS gene expressed in germinated pollen. We discuss the implications of these findings for the evolution of the angiosperm pollen tube wall and suggest new avenues of research to clarify the functional roles of CalS in the seed plant male gametophyte.