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Seed dispersal at alpine treeline: an assessment of seed movement within the alpine treeline ecotone JEREMY S. JOHNSON,1, KEITH D. GADDIS,1 DAVID M. CAIRNS,1 AND KONSTANTIN V. KRUTOVSKY2,3,4,5 1

Department of Geography, Texas A&M University, 810 Eller O&M Building, MS 3147 TAMU, College Station, Texas 77843 USA €sgenweg 2, D-37077 G€ottingen, Germany Department of Forest Genetics and Tree Breeding, Georg-August University of G€ottingen, Bu 3 Department of Ecosystem Science & Management, Texas A&M University, 305 Horticulture and Forest Science Building, MS 2138 TAMU, College Station, Texas 77843 USA 4 N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkina Street, Moscow 119333 Russia 5 Genome Research and Education Center, Siberian Federal University, 50a/2 Akademgorodok, Krasnoyarsk 660036 Russia

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Citation: Johnson, J. S., K. D. Gaddis, D. M. Cairns, and K. V. Krutovsky. 2017. Seed dispersal at alpine treeline: an assessment of seed movement within the alpine treeline ecotone. Ecosphere 8(1):e01649. 10.1002/ecs2.1649

Abstract. Alpine treelines are expected to advance to higher elevations in conjunction with global warming. Nevertheless, the importance of treeline reproductive patterns and seed dispersal within the alpine treeline ecotone remains unresolved. In this study, we address two research questions at mountain hemlock treelines on the Kenai Peninsula, Alaska: (1) What is the primary mode of reproduction and (2) are seeds leading to recruitment derived from within the local treeline populations or are they arriving from more distant seed sources? To answer these questions, we exhaustively sampled mountain hemlock individuals along a single mountain slope, and genotyped single nucleotide polymorphisms using doubledigest restriction site-associated DNA sequencing. First, we assessed mode of reproduction by determining the proportion of sampled individuals with identical multilocus genotypes that are the product of clonal reproduction. Second, we used a categorical parentage analysis to identify parent–offspring pairs, so that the proportion of treeline reproduction events could be spatially quantified and dispersal distance measured. We identified sexual reproduction as the primary mode of reproduction at our study site. Seedling establishment was characterized by extensive seed immigration and gene flow into the ecotone. The average dispersal distance was 73 m with long-distance dispersal identified as dispersal occurring at distances greater than 450 m. We found that production of seeds within the alpine treeline ecotone is not necessarily a requirement for treelines to advance to higher elevations in response to climate change. The extensive seed dispersal and gene flow into the alpine treeline ecotone is likely sufficient to propel the ecotone higher under more favorable climate. Key words: alpine treeline ecotone; dispersal; double-digest restriction-associated DNA; genotyping-by-sequencing; mountain hemlock; parentage analysis; seed immigration. Received 16 November 2016; accepted 18 November 2016. Corresponding Editor: Debra P. C. Peters. Copyright: © 2017 Johnson et al. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. E-mail: [email protected]

INTRODUCTION

zones in approximate equilibrium with climate, but the temporal resolution of those changes is usually restricted to century-scale analysis (MacDonald et al. 1993, Webb et al. 1993). Now, as future climates are expected to change more rapidly than they have in the past, understanding

Over relatively long periods, vegetation is assumed to be in equilibrium with climate (Cole 2010). Late Pleistocene and Holocene climate changes resulted in the shift of major vegetation

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Two different research methods can be used to investigate dispersal: observational or direct (Nathan and Muller-Landau 2000, Lowe et al. 2004). Observational methods require the investigator to physically capture, tag, and monitor the dispersal of seeds or pollen resulting in low sample size and reduced ability to capture LDD events. On the other hand, direct methods using genetic markers and parentage analysis allow a researcher to identify the physical distance any established individual traveled from its paternal and maternal source by comparing the individual’s genotype to that of the likely parents within a given area and identifying a parental match. The task is complicated by the need to generate a complete survey of all individuals contributing genotypes into the area of interest. It requires the assumption that the maternal, seed parent is sampled and that all parental contributors are still alive, meaning that the assumption of exhaustive sampling is often unrealistic (Oddou-Muratorio et al. 2003). This makes the process of parentage analysis challenging for high-density populations, or large geographic areas, often requiring the use of restrictive assumptions. A commonly used set of assumptions was developed by Dow and Ashley (1996). They argued that depending on the number of parental candidates matched to an identified offspring, the seed parent could be assigned based on geographic proximity. For example, if only one parent was identified in a parentage analysis, then the identified parent would be classified as the seed parent given that seed dispersal is generally more limited than pollen dispersal (Oddou-Muratorio and Klein 2008). Additionally, when no parent is identified from within the sampled area, the establishment of a seedling is assumed to be the product of seed dispersal from beyond the sampled area. And not considering type II error, this provides an estimate of seed immigration and can suggest LDD (Bacles et al. 2006). Dow and Ashley assumptions have been used to interpret seed dispersal studies in both temperate (Dow and Ashley 1996, Bacles et al. 2006, Piotti et al. 2009) and tropical species (Arendt 2015), and while imperfect, they do provide insight into dispersal processes. The direct approach has been used to understand dispersal in a variety of environments including both animals and plants in terrestrial (Dow and Ashley 1996, Lian et al. 2008, Piotti et al. 2009, Listle and

the migration potential of species is essential for conservation planning and land management. In some cases, ecosystems may become severely restricted in spatial extent or disappear altogether. For instance, the alpine tundra is in danger of range contraction and diversity loss as subalpine forests migrate upslope (Jump et al. 2012, Greenwood and Jump 2014). Recent research has shown that not all treelines respond in the same way to a warming climate (Harsch et al. 2009, Harsch and Bader 2011, Mamet and Kershaw 2012, Cieraad and McGlone 2014, Chhetri and Cairns 2015). In a tightly coupled vegetation–climate system, we would expect that the treeline should track climate relatively closely over periods as short as a decade (Danby and Hik 2007, Stueve et al. 2011). Unlike animals that can respond quickly to changing environmental conditions, plants are, in most cases, fixed in space and must respond inter-generationally. Therefore, the rapid response of the treeline to changing climate depends on the ability of seeds dispersing beyond treeline, yet it remains unclear whether recruitment of seedlings above treeline is fed by local or lower-elevation seed sources €rner 2012). We understand some of the spatial (Ko characteristics of short-distance seed dispersal as well as some of the characteristics of the shape of the dispersal curve near source trees (Schupp and Fuentes 1995, Greene and Johnson 1996, 1997, Greene and Calogeropoulos 2002, RobledoArnuncio and Garcıa 2007, Oddou-Muratorio and Klein 2008). For instance, gravity- and winddispersed seeds tend to accumulate near the seed source resulting in a dispersal kernel that is leptokurtic. What remains more difficult to understand are those less frequent long-distance dispersal (LDD) events that account for the 99th percentile of a dispersal kernel (Nathan 2006), which we refer to as LDD. Reid’s paradox (Clark et al. 1998) codifies the idea that LDD is necessary for rapid responses of temperate tree species to climate change, based on the observation that rapid migration rates documented for tree species in the paleoecological record are far too high to have been produced by the traditional thoughts on tree life history and restricted dispersal. Despite the revelation of its importance, LDD still remains very challenging to quantify due to the difficulty in observing a LDD event or identifying an individual that is the product of such an event. ❖ www.esajournals.org

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however, difficult to observe and quantify LDD of seeds alone, and assessing the successful dispersal of seeds plus their establishment (known as effective dispersal (Cain et al. 2000, Nathan and Muller-Landau 2000)) is much easier and perhaps of greater biological importance (Nathan et al. 2003) within the context of population growth and spread. If we can quantify where seeds are coming from and how far they are traveling to establish at the alpine treeline, we can assess the likelihood that treelines are strong bioindicators of climate change (Smith et al. 2009) as well as assess the ability of treeline to migrate in a rapidly changing climate.

Reisch 2012, Weinman et al. 2015) and aquatic systems (Markwith and Scanlon 2006, 2007, Hauser et al. 2011). In her review, Ashley (2010) identified that LDD of both seed and pollen in forest trees was more common than previously thought; however, seed dispersal was more limited than pollen dispersal. In many plant species, the mode of reproduction (sexual or clonal) is dependent on the level of abiotic stress in the system (Beerli and Felsenstein 2001). For instance, sexual reproduction (via pollen transfer and seed production) is the dominant mode of regeneration in conifers under climatically and environmentally optimum conditions (Viktora et al. 2011), but under suboptimal conditions, asexual regeneration occurs through layering (Arno and Hammerly 1984), where lower branches adventitiously sprout roots when they come into contact with the soil, eventually separating into an autonomous individual. When seedling viability is reduced due to short growing season and low temperature, as we see at alpine treelines (Harsch et al. 2009, Payne et al. 2011, €rner 2012), vegetative layering functions as an Ko adaptation that improves recruitment (Holtmeier 2003). Nevertheless, these alternative reproductive modes allow for vastly different rates of movement, as new establishment in vegetative reproduction is restricted to areas immediately adjacent to parent trees. Thus, this difference influences the ability of a given species to track climate change along its habitat borders. Given the environmental restrictions at alpine treelines, the relative contribution of sexual and asexual reproduction is uncertain. A subalpine treeline study of black spruce (Picea mariana) identified that the mode of reproduction was mixed sexualclonal using genotype matching (Viktora et al. 2011). Nevertheless, other studies in alpine treelines are few; thus, it is unclear whether this study represents a general pattern or an anecdote. Understanding and quantifying seed dispersal and reproductive mode at treeline is of interest, particularly in the context of understanding global processes and species responses to climate change (Johnson et al. 2016). In most cases, anemochorous tree seeds travel only tens of meters (Howe and Smallwood 1982, Bullock and Clarke 2000), giving rare LDD events a disproportionately higher impact on population demographics when it occurs (Hampe 2011, Carlo et al. 2013). It is, ❖ www.esajournals.org

Objective This research is aimed at addressing the following two questions: (1) What is the primary mode of reproduction in the treeline-forming conifer mountain hemlock (Tsuga mertensiana) and (2) are mountain hemlock seedlings derived from local treeline stands, or are they arriving from more distant seed sources? To answer these questions, we exhaustively sampled mountain hemlock individuals along a single mountain slope on the Alaskan Kenai Peninsula, genotyping DNA single nucleotide polymorphisms (SNPs) identified using double-digest restriction-associated DNA sequencing (ddRAD-Seq). First, we assessed mode of reproduction by determining the proportion of sampled individuals with identical multilocus genotypes that are the product of clonal reproduction. Second, we used an exclusion-based parentage analysis to identify parent–offspring pairs so that the proportion of treeline reproduction events can be spatially quantified and dispersal distance measured. In addition to the parentage analysis, we used a spatially explicit maximum-likelihood neighborhood model to estimate the amount of seed and pollen immigration at the study site and assess the shape of the tail of the dispersal curve. These findings help elucidate the degree to which treeline-forming species can respond to rapid climate change.

MATERIALS AND METHODS Study species—mountain hemlock Mountain hemlock is a monoecious, windpollinated species that has long-distance seed dispersal capacity (Johnson 2016) and an outcrossing 3

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mating system (Owens and Molder 1975, Means 1990, Ally et al. 2000). The species is found principally in wet cool environments on the western coast of North America, extending from the Alaskan Kenai Peninsula to the Sierras of northern California (Means 1990, Taylor 1995). Mountain hemlock is the dominant conifer within the subalpine zone of the Kenai Mountains and is part of the “spruce hemlock zone” (Miller and Walton 2014) consisting of Sitka spruce (Picea sitchensis) and white spruce (Picea glauca). Mountain hemlock reaches sexual maturity and begins producing cones between 20 and 30 years of age (Means 1990). Seeds are primarily winddispersed, with mast year cone crops capable of producing 215,000 to 4,144,000 seeds/ha (Means 1990). At alpine treeline in Alaska, mountain hemlock stands can occur in open parkland form, and above treeline, the species may take on a krumholz form. Vegetative reproduction, through layering, does occur in the krumholz form in Alaska (Means 1990) though it is unknown how common this form of reproduction is. Subalpine mountain hemlock growth is negatively correlated with spring snowpack depth and positively correlated with summer growing season temperature (Taylor 1995, Peterson and Peterson 2001), with warm July temperatures resulting in increased seed production (Woodward et al. 1994).

by 0.2°C per decade over the past 70 years (based on temperature data from the Homer Airport climate station). These changes in temperature have resulted in both an increase in Kenai Peninsula treeline elevation on cool, moist, north-facing slopes and an increase in density of the treeline (but not an increase in elevation) on other aspects (Dial et al. 2007). Similar patterns of treeline response to climate change have been observed at other locations in Alaska (Lloyd et al. 2002).

Sampling We sampled all mountain hemlock individuals along an 860-m transect that extends from the highest mountain hemlock individual on the slope down to the valley floor (between 880 and 610 m a.s.l.; Fig. 1). We observed no evidence of human disturbance along the transect. All individual stems within 300 m of the transect center were collected. The next closest patch of mountain hemlock to the study site was approximately 400 m away from the closest tree on the transect. This isolated patch was chosen because of its spatial characteristics to ensure that we would not fail to sample a potential living seed parent less than 400 m from any given tree. We collected 163 mountain hemlock individuals along the transect. We collected foliage from each tree (approximately 15 cm long branch tips with young needles attached) for DNA extraction, and placed the needles in a plastic zip-lock bag with silica gel for preservation (Colpaert et al. 2005). We stored the bags at approximately 10°C in the field until they were shipped back to the laboratory. We recorded the geographic location of each sample using a handheld GPS (3 m). Upon return from the field, the samples were placed in a 20°C freezer to await DNA extraction and sequencing. In order to determine the age of each sampled tree, we collected a basal tree-core, using an increment borer, from all sampled trees with a basal diameter >5 cm, and a stem cross-section for trees smaller than 5 cm basal diameter.

Study area—Palmer Creek Drainage: Kenai Peninsula, Alaska We based our study along a west-facing elevational gradient, from alpine treeline down to the valley floor in the Palmer Creek drainage of the Chugach National Forest on the Kenai Peninsula, Alaska. The study area is located in the north central portion of the Peninsula in the Chugach–St. Elias Mountains ecoregion (Nowacki et al. 2001) at about 60°470 37″ N and 149°320 11.35″ W. The vegetation structure is typical of the Kenai Mountains and is composed of shrub communities at the lower portion of the ecotone, primarily willow (Salix sp.) and alder (Alnus sp.), embedded in a matrix of bluestem (Calamagrostis sp.) with white spruce and mountain hemlock occurring as the dominant conifer species. Mountain hemlock dominates the alpine treeline ecotone, which then transitions into alpine lichen tundra at approximately 800 m a.s.l. Average summer temperatures (1940–2010) on the Kenai Peninsula have increased ❖ www.esajournals.org

Genome-wide marker development We used a genotyping-by-sequencing (GBS) approach, ddRAD-Seq (Peterson et al. 2012), to genotype all individuals on the transect. We processed tissue by first grinding approximately 30 mg of needle tissue, and extracted genomic DNA following a modified Cetyl 4

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Fig. 1. Individual mountain hemlock (Tsuga mertensiana) locations along the study transect in the Palmer Creek drainage of the Chugach NF on the Kenai Peninsula, Alaska. Individuals were sampled from the valley bottom to the highest mountain hemlock individual. Individual parent–offspring connections identified using parentage analysis are indicated by thick black lines with arrows indicating the direction of movement. The box on the transect map indicates the extent of the inset map. In the one instance where both pollen and seed parents are identified, the red line (pollen) and blue line (seed) are used to distinguish them. The ages of the trees are represented by circles. Large circles represent the oldest trees and smaller circles progressively younger trees.

trimethylammonium bromide (CTAB) protocol (Doyle and Doyle 1987). To develop genome-wide markers, we targeted approximately 1% of the mountain hemlock genome using the restriction enzyme (RE) pair SphI-MluCI. Using the selected RE pair, we generated paired-end (PE) sequencing libraries with ~350 bp long inserts from all 163 ❖ www.esajournals.org

mountain hemlock samples and sequenced them with 150 9 2 cycles in a single HiSeq 2000 lane (Illumina, San Diego, California, USA). This approach allowed us to simultaneously sequence and identify genome variants across all sampled individuals. Using PE sequencing allowed us to improve our alignment success by sequencing 5

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both forward and reverse template strands of the DNA fragments. All library preparation and sequencing was carried out at the University of Texas Genomic Analysis and Sequencing Facility. We used standard GBS bioinformatics analysis performed by the Texas A&M Institute for Genome Sciences and Society. This analysis assessed sequence read quality, de-multiplexed samples, de novo aligned reads into contigs, and identified genomic SNP variants using the dDocent pipeline (Puritz et al. 2014). We quality-filtered the SNPs using VCFtools (Danecek et al. 2011). We filtered the SNP dataset by selecting a 109 minimum coverage depth cutoff and Phred quality score >30. We discarded SNP loci that were not in Hardy– Weinberg equilibrium (HWE) at P < 0.05, that failed to genotype in greater than 95% of individuals, with a minor allele frequency less than 15%, that were not bi-allelic, and that with expected heterozygosity (He) less than 0.2. Individuals missing greater than 15% of the identified SNPs were also discarded. These filters were chosen specifically to identify a set of polymorphic loci that would improve kinship estimation and reduce error in parentage assignment. We choose SNPs that were mostly selectively neutral to improve our estimation of demographic parameters. Currently, there are no standard filtering pipelines, and selection of informative markers depends on the questions being asked. However, a set of guidelines is beginning to be developed in order to reduce bias and error associated with marker selection (Narum et al. 2013).

from the curvature and width of the innermost ring (Applequist 1958). In most cases, we did not add more than 5 yr when estimating establishment date allowing us to bin trees into five-year age classes. We created histograms to investigate the age structure of the transect, and disambiguate parent–offspring relationships in our parentage analysis.

Clone identification To determine the major reproductive pattern within our study site, we compared individual genotypes among our sampled trees. Using the computer program CERVUS 3.0.3 (Marshall et al. 1998, Kalinowski et al. 2007), we conducted an identity analysis, where multilocus genotypes are compared to find near-identical matches, which we labeled as clones. We added nine additional mountain hemlock individuals, sampled from across the Kenai Peninsula, to our trees from the study site as an outgroup to quality-check our identity analysis. We allowed for a 10% mismatch of loci in the multilocus genotypes based on estimated sequencing error and missing genotype information. We classified individuals with unique multilocus genotypes as the product of sexual reproduction rather than clonal reproduction. We further tested the relationship of individuals to each other by estimating kinship (R; Albert et al. 2011). If individuals are clones, then they should have R values of 1; if they are full siblings, then R values would be close to 0.5; and if they are half-siblings, then R values would be near 0.25. Kinship values of 0 indicate no relationship. This analysis was conducted using SPAGeDI 1.4 (Hardy and Vekemans 2002).

Age structure analysis To assess the age structure along our transect, we measured tree age from our collected increment cores and cross-sections following Lafon (2004). We dried cross-sections and cores in an oven for a minimum of 24 h at 100°C and then sanded them with progressively less abrasive sand paper (80 to 400 grit) to draw out the cellular structure of the annual rings (Fritts 1976). We developed a master chronology (Stokes and Smiley 1968) using 23 of the longest cores collected along the transect. This allowed us to identify significant marker rings that facilitated visual crossdating. We dated the remaining increment cores and cross-sections under varying magnification with a stereomicroscope. For any core not showing the pith, we estimated establishment date ❖ www.esajournals.org

Parentage analysis To determine the dispersal pattern at mountain hemlock treeline, we used parentage analysis to identify dispersal and recruitment by excluding all but the most likely parents based on the multilocus genotypes of the candidate offspring (Manel et al. 2005, Jones et al. 2010). The general principle in diploid organisms is that an offspring and a parent will share at least half of their alleles with each other at a single locus (Jones et al. 2010). In our analysis, we allow for mismatches of up to 10%, meaning that parent–offspring relationships can be ruled out after the tolerance threshold has been met. Because of the substantial field and laboratory 6

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dispersal events to be identified and included in the analysis (Pluess et al. 2009). We assigned seed parent status following the assumptions outlined by Dow and Ashley (1996). When only one parent was identified, we assigned it as the seed parent. When we identified both likely parents, we assigned the parental candidate that was geographically closer to the offspring as the seed parent and the more distant parent as the pollen parent. When no parent was identified within our study site, we assumed that the seed was dispersed from beyond the surveyed area, which we defined as LDD. In cases where a seed parent is matched, we calculated dispersal distance based on the Euclidean distance between the parent and the offspring using ArcGIS 10.1 (ESRI 2011). When we found a match between adults, we determined which of the pair was parent and which offspring based on the age of the two sampled trees with the older stem (based on treering-derived ages) being considered the parent. Conservatively, mountain hemlock reaches sexual maturity at about 20 years of age (Means 1990). Thus, for a parent–offspring assignment to be validated, the minimum difference in age between the two candidates must be greater than 20 yr. Dow and Ashley assumptions can create a bias in interpreting the results of parentage analysis because of the difficulty in separating the role of pollen from seed in the respective offspring when using bi-parentally inherited markers (e.g., nuclear DNA SNPs). However, we believe that the general principles of seed and pollen dispersal do allow for meaningful interpretation, and in most cases, the assumptions are reasonable (Oddou-Muratorio and Klein 2008) and provide insight into this life history stage of treelines. As an additional measure of assessing seed movement and immigration within the study site, we employed a maximum-likelihood neighborhood model implemented in NM+ 1.1 (Chybicki and Burczyk 2010). NM+ allows for the assessment of seed and pollen immigration and patterns of gene flow within a spatial neighborhood. This approach uses spatial locations of mountain hemlock at the study site to estimate patterns of mating and average seed dispersal distance within the sampled neighborhood. Because this method does not require a complete set of parent–offspring assignments, it can be used to estimate the degree

efforts involved in sampling large areas or highdensity stands, parentage analysis is often limited to study sites where all potential parents can be sampled in a cost-effective manner. This approach reduces the likelihood of falsely assigning a parent to a sampled individual (type I error), or failing to assign the actual parent within a study area while sampling (type II error). Because of these logistical constraints, sampling is generally restricted to lowdensity species within manageable study areas (Holderegger and Wagner 2008). Although mountain hemlock can be locally gregarious on the Kenai Peninsula, we chose a study site with limited local density where we could exhaustively sample living individuals along the slope. We conducted our analysis using the computer program CERVUS 3.0.3 (Marshall et al. 1998, Kalinowski et al. 2007), which employs a categorical allocation approach based on a maximum-likelihood assignment tests to identify the most likely parent or parent pair from multilocus genotypes, useful when neither candidate parent is known a priori (Jones et al. 2010). In order to determine the confidence of parentage assignments in our dataset, we calculated critical values of likelihood ratios using parentage simulation based on the allele frequencies in our SNP dataset. We simulated 10,000 offspring and 172 parents; 163 parents represented from the transect were assessed and we included an additional nine individuals sampled and sequenced from other locations on the Kenai to provide a secondary estimate of false parental assignment. We assumed that 10% of candidates were sampled and a 10% error rate in likelihood calculations was based on our estimate of sequencing error. Parent–offspring assignments were assessed using the logarithm of the odds (LOD) score. LOD scores were assigned to every single parent–offspring combination along the transect. We estimated the critical LOD scores, used to assess statistical significance, using the simulated parentage analysis. We assigned offspring to parent or parent pairs by matching those individuals with the highest LOD score above the critical threshold. Confidence was assessed at strict, 95%, and relaxed, 80%, confidence levels as is commonly reported in studies using CERVUS. 95% confidence minimizes a wrong parent assignment allowing only the most likely parent to be identified, while 80% confidence increases the likelihood of type I errors, but allows for a larger number of ❖ www.esajournals.org

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Age structure analysis

of pollen and seed immigration into the study site, further contributing to our understanding of seed movement at the study site. We estimated immigration parameters from the exponential-power dispersal kernel using a subset of 70 SNP markers. We estimated immigration of seed (ms), pollen (mp), and kernel shape (bs for seed and bp for pollen) to determine whether the tail of the kernel was “fatter” or “thinner” than exponential. A fat tail, bs < 1, indicates that LDD is more likely to occur than in an exponential tail. For our empirical data, we fit normal, lognormal, Weibull, and gamma curves to assess the shape of the kernel and to estimate LDD. The distributions were fit based on the maximumlikelihood approach using the fitdistrplus package in R (Delignette-Muller et al. 2014). We assessed the fit of each curve using Akaike’s information criterion (AIC), whereby the model with the lowest AIC was chosen as the best model. We then estimated the curve parameters from the best fit model and assessed whether the tail of the curve was fat or thin, an indication of the likelihood of LDD. This approach also allowed us to assess the tail of the dispersal kernel (99th percentile) to quantify LDD events following the suggestions of Piotti et al. (2009) and Nathan (2006).

Age class distributions of mountain hemlock within the sample area showed that the population is mainly composed of a mixed age cohort established between 1900 and 2005. A peak of young seedlings has established over the past decade. Additionally, there is a peak of establishment in the early 1940s. The trees on the transect range in age from less than five-year-old seedlings to the oldest stem cored at 264 years of age. The spatial pattern of the cohorts indicates that the treeline ecotone has experienced infilling over the past two centuries with most successful establishment occurring between 700 and 800 m a.s.l. (Fig. 1).

Clone identification We found no indication of clonal reproduction within our sample set. Clone analysis assessed all possible combinations of individual multilocus genotypes within our study site, using the 353 identified SNPs and allowing for 35 (10%) mismatched loci. All 161 individuals had unique multilocus genotypes. Assessment of pairwise kinship resulted in no individuals with R values greater than 0.35. Forty-three pairwise relationships indicated half-siblings (R = 0.25–0.35) and accounted for 0.34% of kinship comparisons. Average relatedness was R = 0.0359 (CI 0.035– 0.036). We conclude that sexual reproduction dominates the mountain hemlock alpine treeline ecotone within our sample area.

RESULTS Genome-wide marker development From 163 individuals sequenced, GBS resulted in 41,057,267 sequencing reads and 50,952 assembled unique contigs with an average per nucleotide read depth greater than 309; 171,019 putative SNPs were identified throughout the mountain hemlock genome using the dDocent (Puritz et al. 2014) pipeline. Quality filtering reduced the number of SNPs to 5140 (Johnson 2016) using VCFtools (Danecek et al. 2011). SNPs with He less than 0.2 were removed and loci not in HWE (P < 0.05) were removed. Additionally, any SNPs failing to be sequenced in more than 5% of individuals were removed. This process excluded an additional 4787 loci and resulted in a dataset of 353 highly informative genomic markers for clone identification and parentage analysis. Two individuals had greater than 15% missing genotype information and were removed from the dataset. One hundred and sixty-one individuals were retained for further analysis. ❖ www.esajournals.org

Parentage analysis Average non-exclusion probabilities were 0.85 for parent pairs and 0.98 for a single parent. Simulation analysis identified critical LOD values of 1.50 for strict 95% confidence and 4.50 for relaxed 80% confidence. Sixty individuals were considered being of reproductive age and classified as parental candidates, and 160 individuals (i.e., all but the oldest tree on the transect) were retained as offspring candidates. Eighteen of the offspring candidates had at least a single compatible parent match within the study area. In one of these cases, two parents were identified within the transect signifying both seed and pollen dispersal events. One hundred and forty-two candidate offspring did not match to a compatible parent within the treeline ecotone. Paired LOD scores ranged from 6.319 to 1.440 at 95% confidence and 1.601 to 4.475 at 80% confidence. 8

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of the dispersal curve. The dispersal curve was best approximated by the Weibull probability distribution function (AIC = 203.611). The Weibull, gamma, and lognormal curves all represented the empirical data well, with only the normal distribution being an unlikely fit (Fig. 3). LDD was quantified at the tail (99th percentile, Nathan 2006) of the Weibull curve as dispersal that occurs at distances greater than 450 m. The shape and scale parameters of the Weibull curve were estimated at 0.782 (0.140) and 64.754 (20.109), respectively. Using the neighborhood model (Burczyk et al. 2006, Scheiner 2016) and a subset of 70 SNPs, we estimated the dispersal parameters for the exponential-power dispersal kernel. The shape parameters were estimated for both seed (bs) and pollen (bp). The shape of the seed dispersal kernel approximated normal and was slightly fat in the tail (bs = 0.998). However, the pollen dispersal kernel was slightly thin tailed (bp = 1.014). Seed and pollen immigration estimates were lower than our estimates from parentage analysis (ms = 0.121, mp ~ 0). Mean within-neighborhood seed dispersal distance was 102 m with pollen dispersing slightly shorter distance at 72 m.

Fig. 2. Frequency distribution of dispersal events.

Nineteen gametes were identified as being produced within our sample area (5.9%), while 301 gametes were potentially produced outside of it: 142 no-parent matches 9 2 (both gametes) + 17 one-parent matches 9 1 (one of two gametes) = 301. The immigration rate of seeds into the transect was estimated at 89%. Within those individuals dispersed as seeds from mothers within the sampled area, 94% had pollen donors outside of the sample area. It is important to understand the direction and distance of dispersal to assess patterns of forest movement. From the 18 identified parent– offspring matches, dispersal events occurred over distances of 1.44 to 326.85 m (mean 73 m; Figs. 1, 2). All four dispersal events less than 10 m from the seed parent indicated upslope dispersal (the offspring was at a higher elevation than the parent). In contrast, 10 (71.42%) of the 14 dispersal events over 10 m were downslope. There is some evidence that seed dispersal is occurring to higher elevations over relatively long distances. Three identified dispersal events occurred by dispersing upslope to higher elevations. The distances dispersed were measured between 50 and 100 m (max. 97.9 m). It is often the case, and our study is no exception that parentage analysis studies in plants identify maximum dispersal distance corresponds with the maximum extent of the study area. In fact, it is possible that with a larger study area dispersal could be observed at greater distances. Despite this fact, we are able to construct probability distribution functions to interpret our study site. We caution that extrapolation to other systems should be considered carefully. Using measured dispersal distance, we fit probability distribution functions to assess the shape ❖ www.esajournals.org

DISCUSSION Our results indicate that reproduction at the mountain hemlock alpine treeline ecotone in the Palmer Creek drainage is driven by sexual

Fig. 3. Weibull, gamma, lognormal, and normal probability distribution functions (PDF) fit to empirical dispersal data assessed for goodness of fit using Akaike’s information criterion (AIC). *The Weibull curve was chosen as the best curve based on AIC = 203.6.

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species limit. The patchy distribution and floristic simplicity of the stand allowed us to exhaustively sample the species. Moreover, the next closest patch of mountain hemlock to the transect was in excess of 400 m, effectively reducing the likelihood that we would fail to sample all living parent candidates less than 400 m in distance. Taken together, this configuration allowed us to assess both the mode of reproduction dominating the alpine treeline ecotone and the degree to which seeds are arriving from within the ecotone or from more distant seed sources. Our conclusions are supported by findings from previous conifer studies that report seed dispersal and immigration at large geographic distances (Robledo-Arnuncio and Gil 2004, Ally and Ritland 2007, Truong et al. 2007, Lian et al. 2008, Piotti et al. 2009). For instance, long-distance wind dispersal of seeds has been reported in hemlock (207.43 m; Ally and Ritland 2007), pine (26.5–817.2 m; Gonzalez-Martınez et al. 2002, pez de Heredia Gonzalez-Martınez et al. 2006, Lo et al. 2015), spruce (344.66 m; Piotti et al. 2009), and fir (236.9 m; Lian et al. 2008). Animaldispersed seeds in oak species have been found to travel >120 m (Dow and Ashley 1996, Moran and Clark 2011, Payne et al. 2011). Additional studies of seed dispersal in oak have shown greater levels of seed immigration (up to 88%), higher than previously thought (Gerber et al. 2014). Other studies have found similar dispersal patterns in other treeline-forming species. Piotti et al. (2009) recovered approximately 11% of parentage assignments in their study of Norway spruce and determined that immigration accounted for over 66% of gametes. Their parentage analysis found extensive gene flow and dispersal at distances in excess of 300 m and LDD was quantified at greater than 1.8 km. An earlier study of gene flow at alpine treeline in mountain birch (Betula pubescens) found extensive gene flow suggesting adequate capacity to track rapid warming (Truong et al. 2007). Few studies have addressed the dominant form of reproduction at the alpine treeline ecotone in conifers. Contrasting our finding is that of Viktora et al. (2011), who found that in relatively stable habitats black spruce reproduction was characterized as sexual, but at treeline reproduction was mixed clonal-sexual. It is possible that the mountain hemlock treelines we studied did not

reproduction facilitated by extensive dispersal capacity and gene flow. Both seed and pollen traveled distances in excess of 450 m, constituting LDD as defined by assessing the 99th percentile of the dispersal curve. It should be emphasized that the characteristics of the dispersal kernel are strongly influenced by the size of the sampling range. This means that maximum distance and respective frequency of dispersal influence observable dispersal distances. Because of this, we are unable to determine an absolute ecologically justifiable value that represents LDD. Within our study site, the dispersal distances are sufficient to introduce seeds from beyond the sampled treeline ecotone, suggesting a mechanism for treeline advance in the face of shifting climate envelopes. The lack of dual parental matches for all but one sampled individual gives a strong indication that both seed and pollen gene flow are high within the study area, further suggesting that a large proportion of treeline establishment events are the result of extensive immigration and gene flow. Our study site represented a complete census of living trees. The spatial characteristics of the site allowed us to efficiently conduct a parentage analysis. Because we only analyzed a single mountain slope, our inferences may be limited when generalizing to other wind-dispersed conifer treelines. However, we believe that the findings here suggest that treelines of a similar nature and dispersal mechanism may also be maintained by moderate- to long-distance seed and pollen dispersal from beyond the treeline ecotone. By assessing the age structure of established individuals, we can conclude that dispersal events primarily contribute to infilling within the alpine treeline ecotone with little evidence of elevation increases in the contemporary era (approximately the last 100 years). These findings are corroborated by earlier research on the Kenai Peninsula (Dial et al. 2007) and correspond with other dendroecological data showing similar patterns of infilling globally (Szeicz and Macdonald 1995, Suarez et al. 1999, Liang et al. 2011, Chhetri and Cairns 2015). There was a pronounced lack of observed seedlings near and above the species limit at our location despite the availability of suitable sites. Our study site provided a natural transect from the bottom of the hill side extending to the highest mountain hemlock individual at the tree ❖ www.esajournals.org

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experience the same degree of stress as those studied by Viktora et al. (2011) and as such have not needed to rely on clonal propagation to persist. Their treeline study area was at higher latitude and elevation within a boreal continental climate.

average distance of 73 m. We found low kinship values between individual trees and no indication of clonal reproduction within our study area. Further, our study site was dominated by seed immigration from beyond the sampled area accounting for a majority of reproduction events within the alpine treeline ecotone. The importance of these findings is twofold. First, it suggests that, at our site, seeds do not necessarily come from within the local stand, suggesting that local production of seeds is not a necessary requirement for treelines to ascend in elevation with the amelioration of low temperatures and harsh climatic conditions at high elevations. Second, we have estimated seed immigration distances within the alpine treeline ecotone providing a starting point at which to assess treeline response to climate change. The methods and approaches set out in this study should be broadly transferable to other treeline sites. Future research should target a broad range of treeline forms to further generalize our understanding of seed dispersal within this environment.

How important is seed production within the alpine treeline ecotone? One of our overall questions in this study was to assess the degree to which seed production within the alpine treeline ecotone is needed in €rner order to maintain or advance treeline. Ko (2012) suggested that local seed production may not be a dominant factor structuring the alpine treeline. Our results corroborate this assertion. The high levels of gene flow via immigration and effective seed and pollen dispersal from beyond the sampling area suggest that, at least in winddispersed species, the ability of the species to advance to higher elevations is not necessarily dependent on the availability of local seed and pollen production. Why do we not then see a gradual increase in treeline position as opposed to the observed infilling? Previous work has suggested that establishment is occurring well above most current treelines, but that frequent disturbance and mortality balance this high establish€rner 2012). ment rate (Slatyer and Nobel 1992, Ko Our age data show that a large cohort of seedlings has established over the past decade, but the age and spatial distribution of older individuals lead us to conclude that a high mortality rate, due to harsh climate conditions, will likely reduce the surviving individuals substantially. Also, it has been suggested that treeline advance cannot be assessed at less than half-century scales due to time lags associated with mortality and age of €rner 2012). As climates reproductive maturity (Ko continue to warm, especially at high latitudes, the mortality rates associated with harsh climates will likely decrease. As climate becomes more favorable, the number of surviving seedlings will increase and we are likely to see an increase in elevation at mountain hemlock treelines on the Kenai Peninsula, Alaska.

ACKNOWLEDGMENTS This work was supported by the National Science Foundation (Grant Number BCS-1333527) and a Texas A&M Institute for Genome Sciences and Society Fellowship to JS Johnson. The open access publishing fees for this article have been covered by the Texas A&M University Open Access to Knowledge Fund (OAKFund), supported by the University Libraries and the Office of the Vice President for Research. We would like to thank Ellen Gass, Trey Murphy, Charles Lafon, and Clint Magill for assistance in the field and laboratory. Logistical support was provided by the USFS Chugach NF. The authors declare that they have no conflict of interests.

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CONCLUSIONS Our study concludes that, in the case of the wind-dispersed conifer, mountain hemlock, reproduction is sexual and that seeds disperse an ❖ www.esajournals.org

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