Analysis of the genetic diversity and population structures of black locust (Robinia pseudoacacia L.) stands in Poland based on simple sequence repeat markers

Climate change is having a significant impact on the geographical distribution of plant species around the world. Most of these changes are related to a warming climate and a decrease in precipitation during the growing season (IPCC 2021). Evidence exists that the viability of European tree species is decreasing and mortality is increasing (Schuldt et al. 2020). In the future, these changes will lead to shifts in the ranges of native and introduced tree species in Europe (Puchałka et al. 2020, 2023; Dyderski et al. 2018). An example of such a species is the black locust (Robinia pseudoacacia L.), whose natural range is North America: Appalachian Mountains, Ozark Highlands and Ouachita Mountains. R. pseudoacacia is considered a drought-adapted tree species in native and secondary distribution that exhibits high morphological and physiological plasticity and also occurs in regions with annual precipitation below 600 mm a⁻¹ (Mantovani et al. 2014; Klisz et al. 2021).

Black locust is one of the most widely cultivated tree species in the world (DeGomez and Wagner 2001). It occurs in at least 35 countries worldwide (Guoqing et al. 2014), most commonly in Europe, except Lithuania, Latvia, Estonia, Denmark, Norway, Finland and the Balkan countries (Dimitrova et al. 2022; Cierjacks et al. 2013; DAISIE 2006), as well as in the Americas, Africa (South Africa, Nigeria), Australia and Indonesia, and Asia. In Europe, black locust has been growing for more than 200 years and its largest distribution area is Hungary, where it currently occupies more than 23% of the forest area (Rédei 2013). Historical records indicate that black locust was introduced to Europe from a limited number of native populations in the northeastern Appalachians (Liesebach and Schneck 2012; Bouteiller et al. 2019). In the 17th century, Robinia seeds cultivated in Paris were spread throughout Europe. The seed orchards that were established sold seeds from trees that were already growing on the continent (Cierjacks et al. 2013). The black locust was introduced to Poland in 1806 (Tokarska-Guzik 2012). It has been planted in forests since 1860. Currently, six selected seed stands are registered, including three selected seed trees, 44 selected trees and two seed orchards (Wojda 2015). Seed stands of this species are projected to continue to increase until the end of 2035 (Program... 2011). Due to its negative impact on biodiversity and forest ecosystem functioning (Langmaier and Lapin 2020), there are no plans to increase its role in forests or expand the range of this species. Black locust is expected to provide material for fast-growing plantations on former agricultural land (Kowalewski 2013).

In recent years, several stands of black locust not covered by the selection programme have been found in Poland, characterised by a unique straight trunk shape. In fact, it is possible to distinguish (at least) two basic stem forms, referred to as the ‘typical’ and the ‘straight stem form’ (shipmast stem form). By far, the most common and predominant form is the ‘typical’ form, which is used to describe a short tree (up to 5 m tall) that often grows twisted and at an angle, with its crown sitting fairly close to the ground. Trees growing in connected stands may also be twisted in different directions (Zajączkowski 2013). It is presumed that trees with straight trunks, in turn, have a genetically determined ability to form straight trunks that have a much greater height above the ground, even when growing in isolation. Under these circumstances, crowns are more regular, branches are less stout and twigs are thinner and less thorny (Zajączkowski 2013). This tall-stemmed form was previously described as a distinct cultivar, namely, R. pseudoacacia L. var. rectissima Raber, although it is now considered only an ecotype. Apart from its visible anatomical characteristics, this ecotype is more resistant to damage by the black locust borer Megacyllene robiniae Forst. and to stem rot caused by Fomes rimosus (Berk.). On the other hand, it produces few seeds, and is therefore usually propagated vegetatively.

Because R. pseudoacacia is a pioneer species, it is usually used to colonise degraded environments with disturbed soil cover structure relatively easily (Ashby et al. 1980). This species spontaneously colonises open wastelands, mining areas, abandoned fields and pastures, or fire-damaged sites (Cierjacks et al. 2013 and Maringer et al. 2012), thus confirming its high invasive potential. Black locust is a valued species in arboriculture (Grünewald 2009 and Böhm 2011) and is used for production of fuelwood, quality wood, fodder, poles and honey (Keresztesi 1988 and Rédei et al. 2002). It has numerous useful properties, including a high relative growth rate and the production of a large biomass of high-density wood that is easy to dry and process and burns well. Its wood properties make it suitable for timber production in fast-growing tree plantations or energy plantations (Halupa and Redei 2013; McKendry 2001). In Hungary, black locust accounts for about 19% of annual timber production (Malvoti 2015).

However, it is also a species that is currently considered invasive in Europe and Asia (Bartha et al. 2008; Nasir et al. 2005; Osada 1997; Raju 199; Everitt et al. 2007; Richardson and Rejmanek 2011) and is one of the most problematic invasive species in many European countries (Kleinbauer et al. 2010). Black locust has characteristics that are interpreted as negative in relation to native species and have implications for reducing biodiversity (Benesperii et al. 2012; Trentanovi et al. 2013). It forms dense patches with only one species relatively easily and displaces other species. The impact of black locust on the diversity of the flora is already well known and is considered to be clearly negative. Many authors (Taniguchi et al. 2007; Vítková and Kolbek 2010; Von Holle et al. 2006; Vítková et al. 2017) report that black locust forms specific communities in which the herbaceous layer differs significantly from that in stands with native tree species. Black locust significantly increases nitrogen levels in the soil (Rice et al. 2004) and increase in nitrogen levels is among the greatest threats to natural vegetation (Hicks et al. 2011). Conditions under the canopy are becoming more favourable for shade-tolerant and nitrophilous species (Dzwonko and Loster 1997; Hruška 1991; Vítková and Kolbek 2010), while most typical oligotrophic and acidotrophic species are disappearing (Benesperi et al. 2012). This species frequently invades drylands and poses a major threat to xerothermic communities (Vítková and Kolbek 2010; Vítková et al. 2017; Hegedusova and Senko 2011; Tokarska-Guzik et al. 2012).

Genetic variation exists within every species and forms the basis for selection and evolution. The analysis of the distribution of genetic diversity in a species provides useful information for conservation programmes and management at the species level (Dąbrowska et al. 2006). Various molecular markers such as randomly amplified polymorphic DNAs (RAPDs) (Dąbrowska et al. 2006, 2021; Mariette et al. 2001), amplified fragment length polymorphisms (AFLPs) (Mariette et al. 2002; Jump et al. 2007) and simple sequence repeats (SSRs) (Mariette et al. 2002; Gonzalez-Martinez et al. 2004; Arif et al. 2010) have been tested for genetic diversity assessment of forest trees. Till now, such studies on genetic diversity of R. pseudoacacia in Poland are not known. In particular, the level of genetic diversity among R. pseudoacacia populations and the nature of available genetic resources were unclear. Microsatellite markers are ideal tools for identifying individuals and studying genetic diversity due to their ubiquity, repeatability, high degree of polymorphism and codominant mode of inheritance (Carletti et al. 2019; Guan et al. 2019; Nasim et al. 2020). Therefore, SSRs are used to study genetic diversity, genetic linkage mapping and ‘fingerprinting’ of many tree species, including R. pseudoacacia (Guo et al. 2017, 2018; Dong et al. 2019). Knowledge of genetic diversity and variation in tree populations is important because its reduction can reduce a species’ plasticity and resilience to biotic and abiotic stresses (Machida-Hirano 2015). Identification, selection and maintenance of genetic diversity within forest tree species is of particular importance due to their long lifespan, which requires adaptation to different environments and climates. Data obtained from genetic studies can be successfully used in breeding programmes for individual tree species (Sethuraman 2018; Hall 2020). In this study, three nuclear microsatellite markers (Rops15, Rops16 and Rops18) were used to analyse the genetic diversity and population structure of seven high-quality populations of black locust in Poland. These markers were used as a molecular tool to infer the origin of a Polish stand by comparing material with plants from seed orchards with clones of Hungarian origin in the Oborniki Śląskie Forest District (PN) and a seed stand in eastern Germany.

Due to the adverse effects of climate change and the energy crisis, the role and importance of black locust have increased in many countries in recent years (Abri 2021). Because of its resilience to water deficits, it is considered a ‘winner’ species in the face of projected climate warming (Dyderski et al. 2018). Many European countries (e.g. Hungary, Germany, Greece, Poland and Turkey) and Asian countries (e.g. India, China and South Korea) have initiated their own breeding programmes for this species (Dunlun et al. 1995; Dini-Papanastasi and Panetsos 2000; Liesebach et al. 2004; Sharma and Puneet 2006; Lee et al. 2007; Böhm et al. 2011; Kraszkiewicz 2013; Szyp-Borowska et al. 2015, 2020). To apply effective methods in a species management strategy, it is necessary to assess genetic differences among provenances and describe patterns of adaptive geographical variation (Sethurman 2018; Alizoti et al. 2022; Guo 2022). Most forest tree species introduced into Europe exhibit high variation within their natural range, and in contrast, the performance and survival of different provenances can vary substantially when planted outside their natural range (Eilmann 2013; Chakraborty et al. 2016; Merceron 2016).

The primary sources for selection and subsequent improvement of desirable traits are genetic resources for breeders. So, their knowledge, evaluation and use are of great importance for further breeding process (Boczkowska et al. 2016; Cuevas and Prom 2020).

The results obtained in this study provide a first insight into the genetic resources of Robinia pseudoacacia for the management of this species in Poland. In Poland, there are plans to further increase the seed base of this species (Programme... 2011), and it is expected that Robinia will provide material for fast-growing plantations on former agricultural land. Due to its usefulness, black locust, when grown on non-forested land (plantations, former farmland, degraded land, woodlots), can be an important source of wood raw material.

European black locust populations are characterised by low genetic diversity (Liesebach and Schneck 2012; Bouteiller et al. 2019; Alizoti et al. 2022), and populations introduced into foreign areas are often subject to founder effects, leading to an additional reduction in genetic diversity (Rijal et al. 2015; Bouteiller et al. 2021). In the present study, the mean Shannon index value was 0.873, which was lower than that reported by Guo et. al. (2022) for the 36 neutral SSR markers (I = 1.302) or those reported by Huo et al. (2009) and Sun et al. (2009) for the AFLP and ISSR (Inter Simple Sequence Repeat) markers, respectively. The level of genetic variability of the populations we studied can be considered relatively low (Ho = 0.28, He = 0.37). These values are lower than those of Guo et al. (2022), where the mean values of Ho and He were 0.551 and 0.608, respectively, or of Lian et al. (2002) (Ho = 0.615, He = 0.773) and Mishima et al. (2009) (Ho = 0.661, He = 0.739). The mean value of Ho for the three loci was higher than He in most populations. Only the population from Pińczów is characterised by a lack of heterozygous genotypes relative to the expected value. The studied populations are characterised by low richness of rare alleles and unique alleles. Moderate population differences among the studied populations were indicated by FST values (0.412). The observed variability was mainly due to differences within populations; AMOVA showed that the genetic difference within populations was up to 63%.

Under most scenarios, climate change is projected to change the distribution of forest types and tree species in all biomes. It can be assumed that the importance of black locust in times of climate change is likely to increase, both due to rising temperatures and the lengthening of the growing season and in terms of the global environmental policy of countries. In some countries, such as Hungary, a breeding programme for this species has existed since the beginning of the 20th century (Keresztesi 1983) and the strong influence of the stand management system has affected the genetic variability of the progeny (Liesebach and Evald 2012).

Structural analysis revealed specific, extremely interesting correlations between Polish populations of R. pseudoacacia. The studied populations were clearly divided into three clusters; but two of them showed similarity to populations from neighbouring countries. A separate group is formed by the population from Strzelce and a seed orchard from Oborniki Śląskie, in which the population from Hungary is represented, and the second group with the population from Mieszkowice in the Szczecin Forest District, the Pińczów and Wołów populations and the population from Germany. This may be of great importance for practice. Perhaps it is worth postulating the separation of two seed regions, southern (similar to Hungarian) and western (similar to German). There is no universally applicable measure for adapting forests to climate change. Forest managers should, therefore, have sufficient flexibility to deploy the adaptation measures most appropriate for their local situations. The knowledge-based use and transfer of well-documented and characterised forest reproductive material can be an effective tool.

In the present study, the first assessment of Robinia pseudoacacia gene resources in Poland was conducted and provided important information for the breeding of Robinia pseudoacacia.