Workpackage 4: The influence of hydrology on sex ratios in dioecious floodplain species Participating partners : University of Cambridge (UK) (1) University of Lethbridge (Canada) (4) Umeå University (S) (6) 3.1 Objectives The overall objective of this workpackage was to study the influence of hydrology on sex ratios in dioecious floodplain species. For the third year, the objectives were (1) to experimentally determine if sexes respond differently to a range of hydrologic regimes in a greenhouse experiment, and (2) to compile, analyse and report the results (D 4.1 and 4.2). 3.2 Methodology and scientific achievement Variation in sex ratios along hydrological gradients were studied in the common floodplain shrubs Salix lapponum and S. myrsinifolia-phylicifolia at five sites in the free-flowing Vindel River and at six sites in the regulated Ume River in Sweden. All individuals of the species were registered along elevational transects perpendicular to the river, extending from the lowest willow to maximum water-level. In the Vindel River, significant spatial separation of sexes was observed at two sites for S. myrsinifolia-phylicifolia and at one site for S. lapponum. In all these cases, females were found at a lower average elevation than males. In the Ume River spatial separation could not be confirmed at any of the sites. To compare sex ratios between the two rivers, 50 individuals of each Salix lapponum and S. myrsinifolia-phylicifolia were sexed along 12 reaches in the Vindel River and 17 reaches in the Ume River. All reaches were situated within the middlelower sections of the rivers. In Salix myrsinifolia-phylicifolia, females were more common than males along all studied reaches in both rivers. There was no significant difference in sex ratios between the rivers . Sex ratios were statistically significantly biased towards females in 51% of the data sets with mean sex ratios biased at 0.6 male to one female. This is consistent with a female-biased sex ratio of 0.4-0.6 in the region as a whole. In Salix lapponum, sex ratios were more variable, but females dominated at all sites except one. To examine the responses of sexes to different hydrologic regimes, a greenhouse experiment was carried out in Cambridge during 2002. In October 2001, cuttings from previously mapped and tagged female and male individuals of S. myrsinifolia-phylicifolia were collected from populations in the Vindel and Ume Rivers. The sampled population in the Vindel River covered an area of about 1000m2 and was situated 10 km upstream from the confluence with the Ume River. In the Ume River, the sampled trees were located along a 60 m long stretch of river, 10 km downstream from the confluence. Cuttings from female and male individuals of S. myrsinifolia-phylicifolia were collected from the Vindel and Ume Rivers in October 2001. After transport to Cambridge, the cuttings were revived, standardised in weight (all cuttings weighed between 14.5 and 17.5 gms), and planted in rhizopods with soil mimicking conditions in their home habitats. The soils were created by mixing 75% gravel with 25% peat and loamy soil (this 25% consisting of 3 parts peat and 1 part loam). 8 rhizopods were used to create 4 experimental treatments (2 rhizopods per treatment). Each Rhizopod had 8 male and 7/8 female cuttings, each in separate growth tubes. No two cuttings from the same parent tree were used in any rhizopod but a similar range of cutting provenances were present in all rhizopod pairs. The two rhizopods receiving the same treatment were considered to be replicates of each other. A total of 19 source trees provided cuttings for the experiment, 10 of which were male and 9 female. All cuttings received an initial two-week period with optimal growth conditions during which water tables were held a few centimetres below the soil surface to give good soil moisture and good soil aeration. Subsequently, the rhizopods were subjected to hydrological regimes that were chosen to represent ranges of waterlevel variation and precipitation in the Vindel and Ume Rivers (Table 1). The experiment ran from January 28thth until May 7th, 2002. During the experiment, the following environmental measurements were made: Soil suction/pressure using tensiometers connected to Campbell data loggers; soil, water and air temperatures using thermocouples and Photosynthetically Active Radiation (PAR). Shoot length measurements were made at two- week intervals during the experiment. After harvesting in May, measurements of shoot dry weight, root length and root dry weight divided into 10cm sections (measured downwards from the base of the cutting) were made. In addition, stable isotopes of oxygen (18O) in leaf water and carbon (13C) in dry leaf matter were used to evaluate water use and photosynthetic characteristics over the course of the experiment. Measurements were made from harvested leaf samples at fortnightly intervals on one female clone and one male clone across the 4 water table treatments. At the end of the experiment, soil samples were collected from two depths in one growth tube in each rhizopod and analysed for pH, EC, % water, % organic matter, % Ca CO3 and particle size. The data were analysed in a full factorial univariate analysis of variance with root and shoot weights at harvest as response variables and hydrologic regime, population origin and sex as predictors. Cutting diameter was included as a covariate. Growth of the willow cuttings were to various degrees determined by all examined. The hydrologic regime had a significant effect on both shoot and root growth. The fluctuating water regime (regime 2) produced significantly higher root and shoot growth than the other three regimes with shoot weights approximately twice as great in regime 2 as in the other three regimes. The population origin also had a significant effect on growth over the course of the experiment. Both shoot and root weights were significantly higher in plants grown from cuttings with source trees in the freeflowing Vindel River than those from the regulated Ume River. The difference between growth of male and female cuttings across the experiment was significant for shoot weight but not for root weight. 3.3 Socio-economic relevance and policy implication During the last century, most larger rivers in northern Sweden have been regulated for hydropower production. This has led to large changes in water-level regimes, in riparian habitats, and in the structure and composition of the riparian vegetation. If willows display a habitat-related spatial variation in sex ratios on floodplains, it is possible that changes in sex ratios have occurred in regulated rivers with potentially serious consequences for reproductive and dispersal processes. 3.4 Discussion and conclusion Numerous studies have shown ecological, morphological, and physiological differences between genders in dioecious plant species. This is usually interpreted as a consequence of different resource demands for male and female reproduction. As a result, the sexes often display different degrees of spatial separation. Along rivers in northern Sweden, species of the dioecious genus Salix dominate the riparian tree- and shrub vegetation. It is not known if and to what degree these species display spatial separation of sexes and how drastic changes in hydrology will influence the spatial distribution of sexes, reproductive and dispersal processes. The field observations on Salix lapponum and S. myrsinifolia-phylicifolia were not unambiguous. Spatial separation of the sexes, with females dominating at the wetter end of the hydrologic gradient, were observed at some sites in the free-flowing Vindel River but not at all in the Ume River. This may possibly indicate that a regulated and short-term fluctuating hydrologic regime in the Ume River prevents spatial separation. There is, however, nothing in our results to indicate that sex ratios on a reach scale would be affected by a regulated hydrologic regime. Data from the greenhouse experiment show a number of interesting trends. The higher growth exhibited by plants grown from cuttings with source trees in the free-flowing Vindel River compared with growth of cuttings from the Ume River suggests that these Vindel cuttings were perhaps healthier or more vigorous at the outset. Further analysis of data on cutting health collected prior to and during the experiment and choice of cuttings for the experiment is required for full interpretation (cuttings were chosen so that even numbers of healthy male and female cuttings were located in each rhizopod and ignored river origin). The higher growth exhibited by cuttings grown in a fluctuating water table regime, regardless of sex and river origin, suggests that good access to water combined with good oxygenation are preferred conditions for cutting growth. However, considerable further analysis of growth data are required. In particular analyses of total root length and root distribution through the sediment profile, by hydrologic regime are required. It is anticipated that root length/root weight ratios will add interesting additional information on growth under different hydrologic regimes. These growth data also need to be analysed in the light of results of soils analyses performed at the end of the experiment. Data from the studies of water use during the experiment using stable isotopes of oxygen (18O) in leaf water and carbon (13C) in dry leaf matter have not yet been fully interpreted. 3.5 Plan and objectives for the next period The draft academic paper on the influence of environmental variation on sex ratios in dioecious floodplain trees will be finalised and submitted within a year.