Evolutionary Trends in Hydrocharitaceae Seagrasses

This paper provides evidences of the evolutionary pathway followed by one of the main groups of marine angiosperms, the Hydrocharitaceae. Current molecular data has confirmed the aquatic origin of these plants. The Hydrocharitaceae group has a cosmopolitan distribution and is well represented in the fossil record in Europe and North America. Morphological and phylogenetic data has shown dramatic differences between the Hydrocharitaceae and the other marine angiosperms. Furthermore, it supports the hypothesis that aquatic monocot ancestors were able to adapt to a continuously changing environment caused by widespread continental flooding in the Cretaceous Period when seagrasses first occur, to a gradual regression of inland seas during the Eocene leading to subsequent adaptation to a completely submerged marine environment within the subfamily Hydriloideae. Mini-review Article Benzecry and Brack-Hanes; ARRB, 9(6): 1-8, 2016; Article no.ARRB.24354 2


INTRODUCTION
Marine angiosperms (seagrasses) are taxonomically confined to 60 species in 13 genera, assigned to five different monocot families within the single order Alismatales, aquatic subclass Alismatidae (alismatids). Seagrasses are known to inhabit the sea worldwide with the exception of Antarctica, representing the utmost adaptive radiation of freshwater plants on Earth [1]. Physical drivers, such as climate change, ocean currents and tectonic events, have been influential in their distribution. To date there are no studies examining patterns of biodiversity change of seagrasses over global or regional scales. According to Barret et al. [2] and Les [3] aquatic vascular plants in general have been reported as having a conservative macro-evolutionary pattern due to their low genetic variability and population differentiation below the species level.
Aquatic monocots have been indubitably present since the early Cretaceous as confirmed by the oldest fossils assigned to this clade, 110-120 million years old [4,5]. Monocot fossil records also confirm the early divergence of seagrasses during this time [5]. Using molecular clocks, the monocots have been dated between 124--141 MYA [6][7][8][9][10][11]. The origin and evolution of monocots and of those especially known to inhabit the seas is an intriguing subject that has evoked several hypotheses; Arber [ Arber [12] outlined very specialized features that were necessary for plants to have in order to exist and reproduce in marine environments. Her ideas were established from plant characteristics at the species level. She also suggested that marine angiosperms were indirectly derived from land plants that became long adapted to aquatic conditions and subsequently adapted further into salt tolerant or even brackish species, leading to completely submerged marine species. Cronquist [13] believed that the origin of monocots was aquatic and that terrestrial monocots are derived from aquatic premonocots. He also suggested that terrestrial monocots then conversely, gave rise, repeatedly, to aquatic groups. Among the new aquatic groups, some progressively adapted to a marine habitat. concluded that seagrasses adaptation to complete submersion into the sea could have followed three separate adaptation linages. This paper will concentrate on adaptations followed by the Hydrocharitaceae representatives.

METHODOLOGY
This study is largely based on a review of the available literature on the aquatic monocotyledons with emphasis on the Hydrocharitaceae Family including the four seagrasses: Enhalus, Thalassia, Halophila and the fossil Thalassites and three freshwater submerged genera: Najas, Nechamandra and Vallisneria; and observations made on our own (in part unpublished) material and results. The hydrocharitacean Floridian fossil seagrass Thalassites parkavonensis was examined for clues that would lead to the evolutionary trends in the Hydrocharitaceae seagrasses following the report of Benzecry [19] which compared the presence of paracytic stomata in members of the Thalassia testudinum (Hydrocharitaceae seagrass) and Vallisneria sp., a close freshwater submerged relative.
References will be made to representatives of the other seagrass families, Potamogetonaceae/ Zosteraceae, Cymodoceaceae, Posidoniaceae and Rupiaceae in order to better understand the evolutionary trends of the order Alismatales.

Molecular Phylogeny
Marine angiosperms polyphyletic origins were confirm by molecular phylogenetic analyses at the family level [20,21]. Genetic studies of the rbcL gene [1,15,17

Morphological Characters
The marine Hydrocharitaceae differ dramatically from other marine angiosperm lineages in relation to the presence of stomata, their pollination mechanisms and pollen morphology as described by Chen, et al. [ . In terrestrial plants, it is believed that these specialized structures are involved in the exchange of gases between the plant and its environment due to the difference in osmotic pressure between the leaves and the roots. In submerged aquatic plants, these same specialized structures allow the plants to extrude water in a liquid form. However, only recently have we begun to understand and identify some of the environmental factors that control stomatal development [32][33][34][35].
The presence of more advanced stomatal types such as the paracytic stomatal complex (in which subsidiary cells that flank the stoma are parallel with the long axis of the guard cells) and the tetracytic stomatal complex (where guard cells are surrounded by four subsidiary cells) are common among freshwater monocots [36] [37] which is also believed to have occurred in areas of coastal margins" [38,39], have shown to contain paracytic stomata (Fig. 1) similar to those of other Hydrocharitaceae species as described by [13,19,36,[40][41][42] and others.
These discoveries as well as the current environmental conditions where seagrasses found may indicate that phenotypic plasticity is operating in seagrasses just as it has in many aquatic and terrestrial plants [33 accurately determine patterns of plasticity and to investigate their ecological and evolutionary implications, we need to better understand the environmental context in which phenotypes are expressed.

Ecological and Environmental Conditions
The Atlantic Coastal Plain of Florida is underlain by a blanket of Miocene and post siliciclastic deposits that overlie a thick sequence of Tertiary carbonates composed of Eocene to Miocene limestone and dolostone. Florida's limestone bedrock is continuously dissolved by moving water on the surface and underground, thus forming its karst topography. groundwater discharges (SGD) and other karst features present today along the Gulf of Mexico coasts and extending below sea level, occurred during a low stand of the Pleistocene sea when the top of the saturated zone stood lower than the bottom of the deepest natural wells [48 Carruthers et al. [51] reported that outflow from submarine springs present both in Mexico and Florida may be influencing nutrient processes within Thalassia testudinum meadows. It is plausible, therefore, that an influx of freshwater in  [37] which is also believed to have occurred in areas of "leaky , have shown to contain 1) similar to those of species as described by These discoveries as well as the current environmental conditions where seagrasses are found may indicate that phenotypic plasticity is operating in seagrasses just as it has in many aquatic and terrestrial plants [33,[43][44][45][46]. To accurately determine patterns of plasticity and to investigate their ecological and evolutionary we need to better understand the environmental context in which phenotypes are

Environmental
The Atlantic Coastal Plain of Florida is underlain by a blanket of Miocene and post-Miocene siliciclastic deposits that overlie a thick sequence of Tertiary carbonates composed of Eocene to Miocene limestone and dolostone. Florida's ntinuously dissolved by moving water on the surface and underground, thus forming its karst topography. Submarine groundwater discharges (SGD) and other karst features present today along the Gulf of Mexico coasts and extending below sea level, occurred ring a low stand of the Pleistocene sea when the top of the saturated zone stood lower than the bottom of the deepest natural wells [48][49][50]. Carruthers et al. [51] reported that outflow from submarine springs present both in Mexico and encing nutrient processes meadows. It is plausible, therefore, that an influx of freshwater in a marine environment can cause a change in the marine plant's hydrostatic pressure, thereby inducing the phenotypic expression of sto Several environmental factors such as light, temperature, dissolved oxygen and ionic concentration can, indeed, influence the expression of anatomical characters in plants. Allsopp [52] and Sculthorpe [53] reported that environmental factors such as water stress or a change in light conditions have induced a heterophyllous switch in freshwater aquatic plants and that those changes are reversible. Experiments by Ueno et al. [54], Bowes & Salvucci [55], Reiskind et al. [56] and Sultan [57], demonstrated that a change from C3 to C4 metabolism can occur within a plant when carbon dioxide is limited in the water. This biochemical change also causes structural changes such as the induction of Kranz anatomy in submerged amphibious plants [58]. Several environmental factors such as light, temperature, dissolved oxygen and ionic concentration can, indeed, influence the expression of anatomical characters in plants. Allsopp [52] and Sculthorpe [53] reported that water stress or a change in light conditions have induced a heterophyllous switch in freshwater aquatic plants and that those changes are reversible. Experiments by Ueno et al. [54], Bowes & Salvucci [55], Reiskind et al. [56] and Sultan [57], d that a change from C3 to C4 metabolism can occur within a plant when carbon dioxide is limited in the water. This biochemical change also causes structural changes such as the induction of Kranz anatomy in submerged

Thalassites parkavonensis Benzecry & (Fossil Leaf sample # 267Ac, Fairleigh Dickinson University Paleobotanical Collection) SEM micrograph of leaf epidermis and paracytic stomata. Stomatal apparatus comprised of two small guard cells (GC) µm each, with prominent poral thickenings surrounded by two large (43µm x 17µm ) reniform subsidiary cells (SC). Scale bar = 10µm
species are vital components of many freshwater habitats that can term exposure to mesohaline conditions have become the main subject of numerous salt tolerance studies [59][60][61][62][63]. Experiments dealing with chronic or sublethal salinity exposure of submerged aquatic vegetation have resulted in physiological changes [64] and population level changes [65]. Larkin et al. [66] studies of Thalassia testudinum revealed low levels of genetic diversity and differentiation among Thalassia populations. However, Hackney and Durako [67] and Kahn and Durako [68] reported that regional environmental differences in Florida Bay have significantly affected trends in the morphology of Thalassia testudinum. A series of temperature and salinity stress tolerance experiments in Thalassia testudinum [68][69][70][71][72] have confirmed the ability of this species to adapt to environmental changes leading to the full modification necessary for a harsh marine environment. The occurrence of stomata in some marine hydrocharitaceans (Thalassia testudinum and Thalassites parkavonensis) growing in "leaky coastal margins" provides evidence for phylogenic pathways in that family, but does not address the subject of the evolution of seagrasses within other families. The fact that stomata were found in only those seagrasses living in close proximity to freshwater intrusions of coastal environments with SGD, implies that freshwater influx is now and possibly was a recurring stressful condition for the plants. Since only Hydrocharitacean seagrasses collected from coastal environments having freshwater intrusions have been described with stomatabearing leaves, it becomes credible that the predisposition (genetic makeup) for stomata is present in the plant, regardless of expression. Furthermore, the occurrence of stomata in an Eocene hydrocharitacean freshwater Hydrilla species [73,74] and the marine Thalassites parkavonensis [37] from a coastal area with SGD demonstrates that it was a character expressed by hydrocharid in similar environments millions of years ago (about 38 MYA) just as it is today.

CONCLUSION
Records indicate that aquatic monocot ancestors were able to adapt to a continuously changing environment caused by the widespread continental flooding during the Cretaceous Period where seagrasses first occurred [4,5] to a gradual regression of inland seas during the Eocene, leading to the subsequent adaptation into a complete submerged marine environment. Hydrocharitaceae seagrasses' aquatic ancestry, as confirmed by molecular studies [1,17,18] and the facts outlined in this paper, further support the idea of a complete adaptation from a fresh water environment to a submerged marine environment within the Subfamily Hydrilloideae.