Genetic and Ecological Comparisons Between Native and Invasive Populations

of Common Buckthorn

Daniel Miller1, Nastassia B. Vlasava2, Elena V. Spiridovich2, Arkadi N. Skuratovich3, and Jan Malysza1

1 University of Minnesota Landscape Arboretum2 Central Botanical Gardens of the Belarus National Academy of Science3 Institute of Experimental Botany of the Belarus National Academy of Science

Central Botanical Gardenof NAS of Belarus

Institute of ExperimentalBotany of NAS of Belarus

Collaboration Between the Minnesota Landscape Arboretum and the Central Botanical Gardens of the Belarus National Academy of Sciences (2013 – 2018)

• Conservation of Biodiversity Seminars and Expeditions in Belarus

• Minnesota Landscape Arboretum hosted scientist from Belarus to study biodiversity conservation and ecological restorations

• Research Topics:• Comparative evaluations of invasive plants of Belarus

and Minnesota• Cranberry plantations invasive plants research and

management in Belarus• Native orchid conservation and research• Genetic and ecological research of cloudberry (Rubus

chamaemorus)

Comparative Evaluation of Invasive Plants of Minnesota and Belarus

Objective: To study invasive plants that are native in Belarus but invasive in Minnesota and visa versa

From Belarus:

• Rhamnus cathartica (common buckthorn)

• Frangula alnus (glossy buckthorn)• Allaria petiolata (garlic mustard)• Lythrum salicaria (purple loosestrife)• Tanacetum vulgare (common tansy)• Cardamine impatiens (narrowleaf

bittercress)

From Minnesota:

• Solidago canadensis (Canadian goldenrod)

• Solidago gigantea (giant goldenrod)

• Bidens frondosa (Devil’s beggartick)• Acer Negundo (boxelder)• Echinosystis lobata (wild cucumber)• Amelanchier spicata (dwarf

serviceberry)

Invasion History of Rhamnus cathartica in North America

• possibly introduced in the late 1700’s for medicinal uses and as a dye plant

• recognized as a superior hedge plant – possibly first used as a hedge in Salem, MA in the 1830’s

• The American Journal of Horticulture in 1867 reported “it is not profusely furnished with thorns; but, as the trunks of the bushes thicken, it will become so dense, that a mad bull could not go through it”.

• Jewell Nursery in Lake City, MN was calling it “a popular hedge plant in 1892; most Minnesota nurseries were offering it by 1909 including Northrup King and Farmer Seed.

Distribution of Rhamnus catharticain North America

Rhamnus cathartica

distribution by MN DNRsurveyMar-2010 to Feb-2018

Distribution of Rhamnus cathartica in Minnesota

Distribution of Native Rhamnus catharticain Europe and Asia

Distribution of Rhamnus cathartica in Belarus

Invasive Traits

•altered phenology – longer growing season than native shrubs

•shade tolerance – but can also grow quickly in open areas

•escape from natural enemies (rodents, insects, diseases, soil microorganisms, etc.)

•previous ornamental use - higher populations near urban areas where it was heavily used as an ornamental

•widely dispersed by birds

Soil Factors

•prefers basic calcareous soils

•altered soil properties – higher % N in leaves, increased litter decomposition rate – changes in the structure of forest floor communities

•alleopathy – the secondary compound Emodin may have alleopathic effects on nearby plants, deter herbivory and affect fruit consumption and digestion by birds

Rhamnus cathartica

vs.Soil pH

pH>7

Relationship to Soil pH in Minnesota

87.2% of samples are found in the areas with pH in the range 5.6-6.00

pH below 5.6 --- 5 cases 5.61-5.80 --- 325.81-6.00 --- 436.01-6.2 --- 10Above 6.20 --- 5

Relationship to soil pH in Belarus

Populations in the study from USA Middle west and European area of R. cathartica distribution

Minnesota – 2 populationsMichigan – 1 population

Belarus – 3 populations

Comparative Characteristics of Plant Communities

Site

Characteristics

Belarus Minnesota Michigan

forest types Scots pine-Norway

spruce-black alder-silver birch

Sugar maple-American

basswood-red

oak-ironwood

Maple-basswood-oak-hic

kory

soils Sandy, thin liter layer Clay loam sandy loam

abundance of

buckthorn

Sparsely scattered Site 1 (untreated) = 90% of

shrub layer;

Site 2 (restored) = 15%

Site 1 (untreated) = 25%

of shrub layer

Site 2 (restored) =15%

other shrub layer

species

Spindletree (Euonymus

verrucosus), glossy buckthorn

(Frangula alnus), commom

plum (Prunus domestica),

mountain ash (Sorbus

aucuparia), grape woodbine

(Parthenocissus vitacea),

dwarf serviceberry

(Amanlanchier spicata), red

raspberry (Rubus idaeus)

Green ash (Fraxinus

pennsylvanica), Tatarian

honeysuckle (Lonicera

tatarica), riverbank grape

(Vitis raparia), eastern red

cedar (Juniperus

virginiana), Virginia

creeper (Parthenocissus

quinquefolia)

Amur honeysuckle

(Lonicera maackii), white

ash (Fraxinus alba), black

maple (Acer nigra), black

cherry (Prunus serotina),

wild yam (Diascorea

vilosa), tree of heaven

(Alianthus altissima)

No of species in

the ground layer

Site 1 = 73;

Site 2= 58

Site 1 = 5;

Site 2 = 35

Site 1 = 12;

Site 2 = 22

Genetic differentiation of individuals R. cathartica in the regions of natural distribution (Belarus) and

secondary naturalization (Minnesota)

Electrophoresis and fragment analysis of the products of amplification genomic DNA of R. cathartica individuals from several Minnesota locations (TR, TLR), Michigan (NM) and Belarus (RLB, GR) with microsatellite primer RhamA7

145 individuals5 locations2 regions: Belarus, USA2 marker system: SRAP and SSR [Culley and Stewart, 2010] (codominant)

TR19

TL22

Questions we have to answer using spatial genetic variation :

• How native and invasive populations of R. cathartica are fragmented?• Is it similar on both continents?• Can we isolate geographic, environmental or human activity related factors

in R. cathartica nowadays distribution (in USA Midwest)?• What are the conservation implications from integrated analysis of

morpho-ecological and genetic data? • Any possibilities to understand what can control the future effective

spread?• Is it any signs of hybridization and how it can influence further adaptive

evolution of invasive species or the native species distribution?

[Guo, W. Y., Lambertini, C., Pyšek, P., Meyerson, L. A., & Brix, H. (2018). Living in two worlds: Evolutionary mechanisms act differently in the native and introduced ranges of an invasive plant. Ecology and evolution, 8(5), 2440-2452.

Differentiation based on SRAP markers

Figure – Representative

electrophoresis (1.3% agarose) of

products of amplification of

gDNA of individual plants of R.

cathartica (1-5) from Minnesota

locations TR and TLR with

different SRAP primers (A-D). 100

bp и 1 kb – molecular weight

standard (New England Biolabs).

Pop %P N Na Ne I He uHeTL 29.03% 1.935 1.081 1.175 0.158 0.105 0.126(SE) 0.128 0.090 0.039 0.032 0.022 0.026TLR 27.42% 1.468 0.984 1.194 0.166 0.114 0.151(SE) 0.064 0.096 0.040 0.035 0.024 0.032Grand mean (and SE) ovel all loci and locations

28.23% (0.81)

1.702 (0.074)

1.032 (0.066)

1.184 (0.028)

0.162 (0.024)

0.109 (0.016)

0.139 (0.020)

Table – Medium parameters of genetic variability on the population level for Minnesota locations (locations TL и TLR), and also on the species level based on SRAP loci frequencies

Hybrids?Minnesota have native Rhamnus alnifolia (to the north) and the other invasive Rhamnus davurica (to the south)

Primer (loci) Allele, bp Total No of alleles

Populations with private

alleles

RhamA7129, 146, 150, 152, 154, 156, 158, 160, 162, 164, 167, 168, 170, 172, 174

15TL: 129, 172TR: 167GR: 150

RhamD8213, 215, 217, 218, 219, 220, 222, 223, 224, 226

10TR: 213RLB: 218GR: 226

RhamG4

234, 238, 240, 242, 244, 246, 248, 250, 256, 258, 260, 262, 264, 266, 268, 272, 274

17

TL: 238, 258TR: 250, 264RLB: 266GR: 274

Total 42 13

List of codominant loci at R. cathartica individuals from 5 locations in USA (Minnesota - TL, TLR and Michigan - NM) and Belarus (RLB, GR), and private alleles characteristic to the separate locations

Mean Allelic Patterns Across Populations:Conjunction of the GD parameters of populations of R. cathartica from the USA and Belarus and heterozygosity

Minnesota Michigan Belarus

Based on the allelic frequencies of the generated loci, an analysis of the intra- and interpopulation genetic variability of individuals from the studied localities was performed. The following indicators of GD populations were calculated:- The number of polymorphic loci and their percentage; indicators of the total and effective number of alleles (Na and Ne),- genetic fragmentation of populations (Fst), gene flow (Nm), Shannon's genetic diversity (I);- expected, observed and unbiased heterozygosity (He, Ho, uHe), No of private alleles, etc. [Yeh et al., 1999].

Source

Degree of freedom, df

SS* MS Variance component Est. Var.Absolute value %

Among Regions1 10.804 10.804 0.094 4%

Among Pops3 12.390 4.130 0.070 3%

Within Pops140295.620 2.112 2.112 93%

Total 144318.814 2.276 100%

Distribution of Diversity Levels: Analysis of the Molecular Variance (AMOVA) of R. cathartica populations from the USA and Belarus (treated as regions), PhiRT =0.041, PhiPR =0.032 PhiPT= 0.072, P≥ 0.001, at 999 permutations

Principal coordinate analysis of the R. cathartica in Minnesota (TR, TL), Michigan (MN), and Belarus (RLB, GR) showing the relationships based on the pairwise Nei unbiased genetic distance (uNeiP)

Fragmentation between locations by region USA and Belarus: Mean Allelic Patterns Across regions

Pop N Na Ne I Ho* He uHe FUSA Mean 85.0 10.333 4.073 1.631 0.671 0.703 0.708 0.018

SE 0.0 1.202 1.328 0.273 0.054 0.080 0.081 0.156

Belarus Mean 60.0 9.667 3.458 1.597 0.750 0.694 0.700-0.077SE 0.0 1.453 0.619 0.114 0.079 0.048 0.048 0.063

Grand Mean and SE over Loci and PopsTotal Mean 72.5 10.0 3.77 1.61 0.71 0.7 0.7 -0.03

SE 5.6 0.86 0.67 0.13 0.05 0.04 0.04 0.08

Mean values

USA BelarusPopulation

Na 10.3 9.7Na Freq. >= 5% 4.7 5.7Ne 4.1 3.5I 1.6 1.6No. Private Alleles 3.0 2.3No. LComm Alleles (<=25%) 0.0 0.0No. LComm Alleles (<=50%) 0.0 0.0He 0.7 0.7uHe 0.7 0.7

* - hybrid plant was omitted from this data

Principal coordinate analysis of the R. cathartica individuals by region USA and Belarus showing the differentiation and relationships based on the pairwise Nei unbiased genetic distance (uNeiP)

•Differentiation

Conclusions• Species diversity of accompanying plants in the

native R. cathartica habitat (Belarus) was higher than in secondary area of distribution (USA), where restoration sites has higher numbers of species compared to untreated areas.

• Overall USA populations (or region in general) are more diverse by GD indices than Belarus (including private loci and hybrids occurrence)

• Lower fragmentation of USA populations comparing to Belarusian (which are more discrete, Fst)

• No clear fragmentation between regions “USA” and “Belarus” – closer and uniformed material

• Most variation is attributed on the within population level (93%), and only its small part on the among regions (USA/Belarus, 4%) and populations (3%)

• All together data gave us the possibility to predict confirm multiple introductions’ events of R. cathartica in USA.

•Belarusian Foundation for Basic Research 2016-2018 №Б16МС-019

•Special grant of Minnesota Landscape Arboretum.

•Genomic Diversity Lab in University of Michigan

The study supported by

Special Thanks• Belarusian Foundation for Basic Research

• Minnesota Landscape Arboretum

• Genomic Diversity Laboratory, Ecology and Evolution Biology, School of Natural Science, University of Michigan

• Matthaei Botanical Gardens and Nichols Arboretum, University of Michigan

Thanks for your attention!