DOI: 10.5061/dryad.59zw3r2gp
title: Stylidium armeria experimental gene flow data

description: Gene flow can have rapid effects on adaptation and is an important
 evolutionary tool available when undertaking biological conservation and
 restoration. This tool is underused partly because of the perceived risk
 of outbreeding depression and loss of mean fitness when different
 populations are crossed. In this article we briefly review some theory and
 empirical findings on how genetic variation is distributed across species
 ranges, describe known patterns of gene flow in nature with respect to
 environmental gradients, and highlight the effects of gene flow on
 adaptation in small or stressed populations in challenging environments
 (e.g., at species range limits). We then present a case study involving
 crosses at varying spatial scales among mountain populations of a trigger
 plant (Stylidium armeria: Stylidiaceae) in the Australian Alps to
 highlight how some issues around gene flow effects can be evaluated. We
 found evidence of outbreeding depression in seed production at greater
 geographic distances. Nevertheless, we found no evidence of maladaptive
 gene flow effects in likelihood of germination, plant performance (size),
 and performance variance, suggesting that gene flow at all spatial scales
 produces many offspring with high adaptive potential. This case study
 demonstrates a path to evaluating how increasing sources of gene flow in
 managed wild and restored populations could identify some offspring with
 high fitness that could bolster the ability of populations to adapt to
 future environmental changes. We suggest further ways in which managers
 and researchers can act to understand and consider adaptive gene flow in
 natural and conservation contexts under rapidly changing conditions.
description: <strong>Study system</strong> We
 examined F1 hybrid performance of the thrift-leaved trigger plant,
 <em>Stylidium armeria</em>, a species common throughout the
 montane and high elevation woodland areas of southeastern Australia (with
 the current focus on the Australian Alps) (Figure 3). The alpine areas in
 Australia form a rare ecosystem, with treeless alpine vegetation covering
 ~0.15% of the continent, and like other alpine environments around the
 world, they are highly vulnerable to the effects of climate change
 (Hughes, 2003). Cuttings from wild plants were harvested from various
 sites throughout the Victorian and New South Wales high country.
 Outcrossing of the different populations was performed and the F1 progeny
 of these outcrossings were germinated under controlled nursery conditions.
 <em>Stylidium armeria </em>is morphologically variable
 throughout its distribution, with differences thought to be related to
 surrounding vegetation, soil type and climatic factors (Raulings &
 Ladiges, 2001). The pollination unit is a zygomorphic ﬂower, which is
 characterized by the fusion of staminate and pistillate tissues into a
 motile, protandrous column, which is “triggered” when pollinators, usually
 native bees, land on the corolla (Armbruster & Muchhala,
 2009). Twelve populations were sourced, 4 from each of
 3 mountain regions within the Australian Alps (Bogong High Plains,
 Victoria; Mount Buller region, Victoria; and Kosciuszko region, New South
 Wales) to include in the experimental crosses (Table 1, Figure 4). The 3
 regions vary in distance from each other between ca. 50-200 km.
 <em>Stylidium armeria</em> is genetically highly
 differentiated between Victorian alpine regions based on pooled SNP data,
 comparable to differentiation seen for alpine herbaceous plants, which
 tend to be more differentiated than alpine shrubs (Bell <em>et
 al.</em>, 2018). Within each region, the four populations differ in
 elevation by 174-227 m and distance by 3-15 km from each other. Within
 each population, at least 30 plants were collected as cuttings from
 rhizomes, each including a basal rosette of leaves. Each collection
 spanned at least a 100 m<sup>2 </sup>area. Plants were first
 transferred in Fall 2011 after collection into planting tubes with a
 “native mix” soil medium used at Burnley Campus (University of Melbourne)
 glasshouses, which consists of pine bark, peat and sand. After winter,
 plants were transferred individually into 7-inch pots. After pollinations
 were initiated some plants died due to lack of soil drainage, which may
 have contributed to some failed pollinations.
   <strong>Experimental
 crosses </strong> Experimental pollinations
 between all populations, including within population crosses, were
 conducted to examine outcrossing effects of three broad spatial categories
 of gene flow: within site/population (WP), the site of collection; within
 range (WR), between sites within a mountain range; and between mountain
 ranges (BR). Crosses were completed during summer and fall of 2012 (443
 total crosses). Within each population, sire plants were chosen randomly
 during flowering and were mated with up to 2 dams from each population,
 including their originating population. Dams were chosen randomly within
 each population, with replacement. Plants within each population were used
 only once as pollen donors (sires) to individuals in their population and
 to all other populations, but could also serve as pollen receivers (dams)
 to other sires in the study. Two replicate flower pollinations were made
 for each cross. Crosses were completed with equal directionality between
 populations; that is to say, each population served as both pollen donor
 and pollen receiver to each other population. This design produced more
 possible crossing combinations in increasing order of geographic scale: WP
 = 12 possible types; WR = 18 possible types; BR = 48 possible types. We
 used this design to increase sampling size and variety in anticipation of
 outbreeding depression from longer-distance gene flow crosses, and we
 considered WP crosses to serve as a natural control against which to
 compare WR and BR crosses. To test for background or contamination
 pollination, 69 individual flowers, randomly chosen, were marked to test
 for unintended seed set rate. Thirty flowers were randomly chosen and
 self-pollinated to test for self-incompatibility. Of
 the total pollinations observed (including crosses, self-pollinations, and
 tests of inadvertent pollination), 262 yielded seeds (59.1% of 443). Some
 crosses may have been unsuccessful due to plant stress that occurred from
 lack of soil drainage, but this effect was independent of population of
 origin. Capsules were harvested when mature and dried, and the resulting
 seeds were later counted and the seed lots weighed to produce an average
 seed weight (number of seeds divided by total weight). F1 seeds were sowed
 into 7 x 8-cell seedling trays during autumn of 2014. The cells were
 partially filled with the native mix and covered with a layer of “seed
 raising mix,” which consisted of 5 parts medium-grade pine bark, 5 parts
 fine pine bark, 1 part coarse sand, and 1 part sieved peat, including the
 additives Saturaid (1500 g/m<sup>2</sup>) and dolomite (750
 g/m<sup>2</sup>). Each replicate included three seeds sown per
 cell. Depending on seed availability, 1-15 replicates were sown per seed
 family and all were completely randomized across 50 trays included in the
 study, for a total of 2,800 replicates sowed. Following the completion of
 sowing, trays were treated with Regen Smokemaster 2000, a smoke water
 solution applied at a rate of 100 mL per 1L of water, sprayed per square
 meter in order to trigger earlier germination. Trays were randomly rotated
 once weekly, during which trays were monitored for evidence of germination
 and survival. Seedlings (520 total) were transferred into larger pots with
 new potting material after several months’ growth. Plants were grown for
 235 days between the first germinant and the end of the growth trial.
 Plant sizes were estimated by multiplying the longest aboveground height
 and width measurements of surviving plants (493 in total). Plant size was
 also standardized by dividing the final plant size by the number of days
 since germination. Percent germinated plants, seed number, average seed
 weight, final plant size, and the variance in final plant size were all
 compared to assess the outcomes of outcrossing among the three general
 spatial categories, WP, WR, and BR.  
 <strong>Statistics</strong> Our
 main question was, what is the effect of crossing type among the three
 spatial categories? We used means comparison methods (ANOVA–and
 non-parametric Kruskal-Wallis tests by ranks) to test for differences
 among crossing types. We also used generalized linear models (GLM) to
 model additional effects, including the variation of specific crosses
 within WP, WR, and BR categories. Models incorporating all cross types
 simultaneously could not run due to too many missing rows of data (e.g., a
 row with a WP cross type cannot have a BR cross type, and vice versa).
 However, we did test these effects in reduced models, separating WP and WR
 effects from BR effects (see Supplemental Information). Plant size data
 were square root-transformed to meet parametric assumptions and analysis
 of variance (ANOVA) was used to detect significant differences among
 crossing categories. Because transformations of seed number and average
 seed weight data still failed to meet parametric assumptions, we instead
 used Kruskal-Wallis tests to test for differences among crossing types for
 these two variables. Chi-squared analysis was used to test for differences
 in germination. Levene’s test of equality of variances was used to test
 for significant variance differences among crossing types in plant size.
 Finally, we tested for the effect of spatial distance of plant crosses
 using the estimated Haversine distance between populations on the above
 seed and plant size variables. Crosses within populations were assigned a
 distance of 0 km. Because crossing distance data failed to meet
 assumptions of parametric analyses, we used Spearman’s rho
 (<em>ρ</em>) rank correlations to test the effect of crossing
 distance on seed and plant size traits. All analyses were carried out in
 the JMP statistical package (version 16.0.0, SAS Institute, Inc.,
 2021).
description: # Stylidium armeria experimental gene flow
 [https://doi.org/10.5061/dryad.59zw3r2gp](https://doi.org/10.5061/dryad.59zw3r2gp) The dataset contains germination, seed, and plant size data resulting from experimental crosses (pollinations) between populations of the thrift-leaved trigger plant (*Stylidium armeria)* from the Australian Alps. Plants were collected from natural locations, grown in glasshouses until flowering, and cross pollinated as described in the Methods. Seeds resulting from pollinations were counted, weighed, and sown, and the resulting seedlings were raised for several months. Plant sizes were estimated from the longest plant height and width. ## Description of the data and file structure There are three datasets, each contained within a csv spreadsheet. The germination_dataset.csv file contains information about which crosses germinated. Variable names and abbreviations used include the following: sire pop = population from where pollen originated dam pop = population from which maternal plant originated cross identity: specific individual plants involved in cross sire region = mountain region from where pollen originated dam region = mountain region from which maternal plant originated cross type = cross originated from within population, within region, or between region germinated = seeds germinated (Yes = 1) or not (No = 0) Population "STR" = "STL" The seeds_dataset.csv file contains information about the number of seed produced, the mass of seed lots, and the average weight of seeds. Variable names and abbreviations used include the following: sire = individual plant from where pollen originated; codes are population-individual number; e.g., K3-6 is individual 6 from population K3 dam = individual plant from which maternal plant originated; codes are population-individual number; e.g., K3-6 is individual 6 from population K3 \# of seeds = number of seeds produced from pollination mass (mg) = total mass of seeds produced from pollination average seed mass (mg) = # of seeds divided by mass treatment = pollination originated from selfing (S), unpollinated test (T), between-region (B_R), within-population (W_P), or within-region (W_R) cross code = sire and dam involved in cross between-region type = specific regions involved in the cross within-region type = specific region within which cross was made within-population type = specific population within which cross was made cross distance (km) = haversine distance in kilometers between populations Population "STR" = "STL" missing data values for seed mass are blank cells The plant_size_dataset.csv file contains information about plant sizes. Variable names and abbreviations used include the following: sire pop = population from where pollen originated dam pop = population from which maternal plant originated cross code = sire and dam populations involved in cross cross distance (km) = haversine distance in kilometers between populations cross identity: specific individual plants involved in cross within-population type = specific population within which cross was made within-region type = specific region within which cross was made sire region = mountain region from where pollen originated dam region = mountain region from which maternal plant originated cross type = cross originated from within population, within region, or between region between-region type = specific regions involved in the cross days of growth = number of days from seedling emergence to harvest height (mm) = plant height in centimeters width (mm) = plant width in centimeters plant size (mm^2) = height multiplied by width growth/day = plant size divided by days of growth Population "STR" = "STL" missing data values for days of growth and growth/day are blank cells For all sheets, site (population) location information is given in Table 1 of the main article. ## Sharing/Access information All related information can be found in this dataset or within the main article of supplemental information of the article. ## Code/Software All analyses were run in the JMP statistical package as described in the Methods.