Elevation filters seed traits and germination strategies in the eastern Tibetan Plateau

Seeds are the colonizing propagules for many plants and may therefore contribute to the filtering of species during the process of colonization and community assembly. Environmental filtering of seed traits may occur among species and influence community composition, or within species and influence the environmental breadth that a given species inhabits. To test for evidence of such filtering of seed traits, we measured morphological and germination traits of seeds of 408 angiosperm species collected across an elevational gradient in the eastern Tibetan Plateau grasslands. We tested for elevational filtering of traits at the species level, as well as within 22 of those species that occurred at different elevations, in order to test whether within-species variation reflected among-species patterns. Elevational patterning occurred in both seed morphology and seed germination. Seeds were smaller, more elongated, and had a higher surface area:volume ratio and shorter germination times at higher elevation. Seed morphology was associated with germination such that more elongated and smaller seeds with a higher surface area:volume ratio germinated faster, leading to earlier germination in seeds from high elevation. Within species, elevational variation in seed traits was observed in several species, but species differed in how those traits were distributed across elevation. These results suggest that taxonomic differences in seed traits may contribute to elevational variation in the species composition of plant communities, but that seed traits may be variably selected by elevation within species.

Methods
Study region and sites

The study region included the Gannan and Ruoergai located on the north-eastern edge of the Tibetan Plateau in China (100°45'55"–104°45'30"E, 33°35'06"– 35°35'16"N), about 55000 km2 . The climate is mainly alpine with a mean annual precipitation of 532-693 mm. The growing season generally ranges from late April to early November, but only the period from June to August (about 90 days) is free of night-time frost. Mean annual temperature across all sites is 2.2°C (-18.6°C on January and 18.0°C in July).High-elevation sites had lower temperature and higher precipitation than low-elevation sites. The habitat type is primarily alpine meadow, which is dominated by native monocotyledons (such as Poaceae, Cyperaceae) and native dicotyledons (such as Asteraceae, Brassicaceae, Fabaceae, Gentianaceae, Lamiaceae, Ranunculaceae, Scrophulariaceae).

We established 86 study sites across this region at elevations ranging from 1718 m to 4070 m. The sites were distributed along several mountain ranges within areas with low grazing disturbance. The area of each species’ collection site was at least 30,000 m2, in order to obtain at least 1,000 seeds for each species. The elevation of each site was recorded with a GPS instrument.

Seed collection

Across all sites, we identified 408 unique species, most of which were annual and perennial herbaceous plants, but a few were perennial woody plants . 375 species were found in only one elevation, 32 species were found in two elevations, 1 species was found in three elevations, for a total of 442 species x site combinations. Perennials were more prevalent at high elevation than annuals. These species were members of 54 plant families, following the Angiosperm Phylogeny Group Ⅳ (2016). 

Within each site, at least 1000 ripe seeds of each species were collected from at least 20 distinct individual plants of each species in late summer and autumn of 2012. Plants were haphazardly sampled to represent an even distribution across the area of the study site. Seeds were cleaned of appendages (mostly pappus) and stored in paper bags. After air-drying to a constant mass at room temperature (15°C), one-third of the seeds were randomly selected for measurements of morphological traits. The remaining seeds were stored dry at the Research Station of Alpine Meadow and Wetland Ecosystems of Lanzhou University (elevation 2950 m a.s.l, without temperature regulation), the storage temperature was similar to the ambient alpine temperatures. Seeds were stored from the time of collection (late summer and autumn 2012) to the beginning of the germination season in the study area (April 2013). In many alpine and sub-alpine species, dry storage can reduce dormancy and promote germination.

Seed traits

We measured and assessed three morphological traits of seeds: mass, shape, and surface area:volume ratio (SVR). To measure mass, three random samples of 100 seeds were weighed with an analytical balance from each species from each site. The mean of the three measurements, divided by 100, was used as the estimate of individual seed mass that was for analyses.

To measure surface area, 10 randomly selected seeds from each species and site were scanned, and the image was analyzed with Epson Expression 10000XL and imaging software (WinSEEDLE, Canada). The depth of each seed was measured using digital Vernier calipers to enable calculations of seed volume and surface area. The shape of each seed was calculated as the variance of seed dimensions (length, width, and depth), after transforming values so that length is unity. With this metric, perfectly spherical seeds have a value of 0, and elongated seeds have values of up to 0.3. 

Germination experiment

The germination experiment was conducted at the beginning of April in 2013. 50 seeds of a given species and site were incubated on a double layer of filter paper moistened with distilled water in Petri dishes (9cm diameter), with three replicate plates per species (150 seeds per species x site combination). The Petri dishes were placed in temperature chambers with a diurnal fluctuation of temperature (25°C day, 12 h light; 5°C night, 12 h darkness) with a 12-h light and 12-h darkness and a relative humidity of about 70% to reflect natural seasonal temperature fluctuations. This temperature regime was chosen to encompass the temperature range that occurs during the germination period across all elevations. The average temperature decreases with increasing elevation, but the onset of the germination season is later at higher elevation. As a consequence, the temperature range during the germination season are similar at different elevations. The experimental temperature range (5/25°C) was selected to ensure that all species experience a temperature range that occurs within the location of their collection.

Each day, the germinated seeds (radicle protruding from the seed coat) were recorded and removed from the Petri dishes. Distilled water was added to the filter paper as needed. The seed germination experiment lasted 40 days, after which negligible additional germination was observed. If seeds did not germinate by 40 days, the germination time was recorded as a missing value. The viability of un-germinated seeds was tested using the triphenyltetrazolium chloride test (TTC) . Germination traits were calculated using only viable seeds.

Two germination indices were used to quantify germination on each plate: Germination Proportion (GP) was measured as:

GP = n/N

where n = the total number of germinated seeds in a plate, and N = total number of viable seeds in the plate. Germination time (GT) was measured as:

GT =∑ (Gi×i) / ∑ Gi

where: i = number of days between seed sowing (day 0) and seed germination; Gi, number of seeds germinated on day i.

种子是多数植物的定居繁殖体，因此可能在定殖与群落构建过程中参与物种过滤。物种间的种子性状环境过滤可影响群落组成，物种内的此类过滤则会调控特定物种所占据的环境幅宽。为检验种子性状存在此类过滤的证据，我们对青藏高原东部草原沿海拔梯度采集的408种被子植物（angiosperm）种子的形态与发芽性状进行了测定。我们分别在物种水平检验了性状的海拔过滤效应，并针对其中22种在不同海拔均有分布的物种开展种内检验，以探究种内变异是否与种间模式一致。
结果显示，种子形态与发芽性状均存在海拔格局：海拔越高，种子越小、越细长，表面体积比越高，且发芽时间越短。种子形态与发芽性能相关，即更细长、体积更小且表面体积比更高的种子发芽更快，这使得高海拔来源的种子发芽更早。种内层面，多个物种的种子性状均存在海拔变异，但不同物种的性状沿海拔的分布模式存在差异。上述结果表明，种子性状的分类学差异可能驱动植物群落物种组成的海拔变异，而种内的种子性状则可能随海拔受到差异化选择。

方法
研究区域与样地
本研究区域位于中国青藏高原东北缘的甘南与若尔盖地区（100°45′55″–104°45′30″E，33°35′06″–35°35′16″N），总面积约55000 km²。气候以高寒气候为主，年平均降水量532–693 mm。生长季大致为4月下旬至11月上旬，但仅6月至8月（约90天）无夜间霜冻。所有样地的年平均气温为2.2°C（1月平均-18.6°C，7月平均18.0°C）。高海拔样地的温度更低、降水量更高。生境类型以高寒草甸为主，优势类群包括本土单子叶植物（如禾本科Poaceae、莎草科Cyperaceae）与双子叶植物（如菊科Asteraceae、十字花科Brassicaceae、豆科Fabaceae、龙胆科Gentianaceae、唇形科Lamiaceae、毛茛科Ranunculaceae、玄参科Scrophulariaceae）。

我们在该区域沿多条山脉设置了86个研究样地，海拔范围为1718 m至4070 m，所有样地均位于放牧干扰较低的区域。每个物种采集样地的面积至少为30000 m²，以确保每个物种可采集到至少1000粒种子。使用GPS记录每个样地的海拔信息。

种子采集
在所有样地中，我们共鉴定出408个独特物种，其中多数为一年生与多年生草本植物，少数为多年生木本植物。其中375种仅在单个海拔样地被发现，32种分布于2个海拔样地，1种分布于3个海拔样地，总计产生442个物种-样地组合。多年生植物在高海拔区域的占比高于一年生植物。按照被子植物系统发育组Ⅳ（Angiosperm Phylogeny Group Ⅳ, 2016）的分类标准，这些物种隶属于54个植物科。

2012年夏末与秋季，我们在每个样地内从每个物种的至少20株不同个体上采集至少1000粒成熟种子。采样采用随机取样策略，以确保覆盖研究样地内的均匀分布。种子清理掉附属结构（多为冠毛）后装入牛皮纸袋储存。在室温（15°C）下风干至恒重后，随机选取三分之一的种子用于形态性状测定，剩余种子储存于兰州大学高寒草甸与湿地生态系统研究站（海拔2950 m a.s.l.，无温度调控设施），储存温度与周边高寒环境温度相近。种子储存时间为2012年采集后至研究区域2013年4月的发芽季。对于多数高寒与亚高寒物种而言，干式储存可打破休眠、促进发芽。

种子性状测定
我们测定并评估了种子的3项形态性状：单粒质量、形状与表面体积比（surface area:volume ratio, SVR）。
单粒质量测定：从每个物种的每个样地样本中随机选取3组100粒种子，使用分析天平称重，将3次测量的平均值除以100，得到单粒种子质量用于后续分析。
表面体积比测定：从每个物种的每个样地样本中随机选取10粒种子进行扫描，使用爱普生Expression 10000XL扫描仪与成像软件（WinSEEDLE，加拿大）分析图像。使用数显游标卡尺测定每粒种子的厚度，以计算种子体积与表面积。将种子的长标准化为1后，以种子各维度（长、宽、厚）的方差计算种子形状指数。该指数下，完美球形种子的数值为0，细长形种子的数值最高可达0.3。

发芽实验
发芽实验于2013年4月初开展。将每个物种-样地组合的50粒种子置于铺有两层滤纸的培养皿（直径9 cm）中，加入蒸馏水润湿滤纸，每个物种设置3个重复培养皿（每个物种-样地组合共计150粒种子）。将培养皿放置于人工气候箱中，采用昼夜温度波动模式：白天25°C、光照12 h；夜间5°C、黑暗12 h，相对湿度约70%，以模拟自然季节温度波动。选择该温度制度是为了覆盖所有海拔区域发芽季的温度范围。尽管平均温度随海拔升高而降低，高海拔区域的发芽季起始时间更晚，因此不同海拔区域发芽季的温度范围相近。本实验选取的5/25°C温度范围可确保所有物种均经历其采集地发芽季的自然温度区间。

每日记录并挑出已发芽的种子（胚根突破种皮），按需向滤纸补充蒸馏水。发芽实验持续40天，此后未观察到显著的额外发芽。若种子在40天内未发芽，则将其发芽时间记为缺失值。使用氯化三苯基四氮唑测试（triphenyltetrazolium chloride test, TTC）检测未发芽种子的活力，仅统计有活力种子的发芽性状。

我们采用2个发芽指标量化每个培养皿的发芽情况：发芽率（Germination Proportion, GP）计算公式为：
GP = n/N
其中n为培养皿内已发芽种子总数，N为培养皿内有活力种子总数。
发芽时间（Germination Time, GT）计算公式为：
GT =∑ (Gi×i) / ∑ Gi
其中：i为播种日（第0天）至种子发芽的天数；Gi为第i天发芽的种子数。