Scots Pine Forest in Central Europe As a Habitat for Harmonia Axyridis: Temporal and Spatial Patterns in the Population of an Alien Ladybird

FOLIA OECOLOGICA – vol. 47, no. 2 (2020), doi: 10.2478/foecol-2020-0010

Scots pine forest in Central Europe as a habitat for Harmonia axyridis: temporal and spatial patterns in the population of an alien ladybird

Peter Zach1*, Milada Holecová2, Marek Brabec3, Katarína Hollá2, Miroslava Šebestová2, Zdenka Martinková4, Jiří Skuhrovec4, Alois Honěk4, Oldřich Nedvěd5,6, Juraj Holec7, Peter M.J. Brown8, Miroslav Saniga1, Terézia Jauschová1, Ján Kulfan1

1Institute of Forest Ecology, Slovak Academy of Sciences, Zvolen, Slovak Republic 2Department of Zoology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic 3Department of Statistical Modeling, Institute of Computer Science, Czech Academy of Sciences, Praha, Czech Republic 4Crop Research Institute, Praha 6-Ruzyně, Czech Republic 5Biology Center of Czech Academy of Sciences, Institute of Entomology, České Budějovice, Czech Republic 6Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 7Slovak Hydrometeorological Institute, Bratislava, Slovak Republic 8Anglia Ruskin University, Cambridge CB1 1PT, United Kingdom

Abstract Zach, P., Holecová, M., Brabec, M., Hollá, K., Šebestová, M., Martinková, Z., Skuhrovec, J., Honěk, A., Nedvěd, O., Holec, J., Brown, P.M.J., Saniga, M., Jauschová, T., Kulfan, J., 2020. Scots pine forest in Central Europe as a habitat for Harmonia axyridis: temporal and spatial patterns in the population of an alien ladybird. Folia Oecologica, 47 (2): 81–88.

Understanding of habitat favourability has wide relevance to the invasion biology of alien species. We studied the seasonal dynamics of the alien ladybird Harmonia axyridis (Coleoptera: Coccinellidae) in monoculture Scots pine forest stands in south-west Slovakia, Central Europe, from April 2013 to March 2015. Adult H. axyridis were collected monthly across seven randomly selected pine stands of different ages and canopy closure, from the lower branches of pine trees, and larvae were recorded qualitatively. Adults were recorded all year round, most abundantly in November and least abundantly in February. The relationship between the abundance of H. axyridis and selected forest stand characteristics was modelled using the negative binomial Generalized Additive Model with penalized spline component in month (seasonality) effect, year, canopy closure and age effects and the random effect of forest stand (sample area effect). The abundance of H. axyridis was significantly influenced by the age of stand and seasonality (with month granularity) for both closed and open canopy stands, whereas the effects of canopy closure and sample area were not significant. The bimodal pattern of seasonal dynamics of H. axyridis on Scots pine was common for closed and open canopy stands, with two peaks reflecting the cyclic movement of the species from and to overwintering sites. Harmonia axyridis utilized certain pine stands preferably for foraging during the growing season and certain stands for refuge during winter. The ladybirds were found in highest numbers in the 15 year old closed canopy stand (overwintering site). The occurrence of both adults and larvae in most stands indicated a suitability of Scots pine forest for ladybird breeding. The model of year-round dynamics of H. axyridis has been presented for the first time within the invaded range of the ladybird in Europe.

Keywords harlequin ladybird, invasive insect, invaded range, Pinus sylvestris, seasonal changes

81 Introduction iferous trees enable the study of H. axyridis all year round. Despite this, coniferous trees within Europe have not been The harlequin ladybird Harmonia axyridis (Pallas) systematicallly studied throughout the whole year as hosts (Coleoptera: Coccinellidae) is the most invasive ladybird of H. axyridis. Brown and Roy (2018) surveyed H. axyridis on Earth (Majerus, 2016) originating from East Asia on Scots pine from April to October, and Holecová et al. where it has a wide range (Orlova-Bienkowskaja et al., (2018) from October to March. 2015). Within this range, specifically in Japan, H. axy- The Scots pine is the most widely distributed species ridis is mainly associated with trees and shrubs (Osawa, of pine in the world, extending from Europe to northern 2000) and mostly occurs in agricultural fields, orchards, Manchuria and the sea of Okhotsk (Critchfield and Lit- parks, residential yards and gardens as well as in decidu- tle, 1966; Rehfeldt et al., 2002; Barna et al., 2020). Our ous and coniferous woodlands but it is uncommon in nat- study provides insights into the year-round dynamics of ural forests (Osawa, 2011; Noriyuki and Osawa, 2015, H. axyridis in monoculture Scots pine forest in south-west 2016). This habitat generalist and predator with a broad Slovakia (Central Europe). Harmonia axyridis was first prey range (Noriyuki et al., 2011, 2019) is also found on recorded in Slovakia in 2008 (Majzlan, 2008; Panigaj et pine trees (Noriyuki and Osawa, 2016). In the invaded al., 2014) and in the surveyed Scots pine forest in 2012 (M. range within Europe, H. axyridis is also a habitat general- Holecová, unpublished data). Over the period April 2013– ist (Adriaens et al., 2008; Roy et al., 2016; Honěk et al., March 2015 we sampled Scots pine trees to (1) investigate 2018) occurring frequently in urban areas where it is often changes in the densities of H. axyridis in Scots pine stands the most abundant (dominant) ladybird on lime (Tilia spp.) systematically all year round, (2) examine the effects of se- and maple trees (Acer spp.) (Roy et al., 2016; Viglášová lected stand characteristics (variables), including tree age et al., 2017; Brown and Roy, 2018; Honěk et al., 2015, and canopy closure, on the densities of H. axyridis, and (3) 2019). The ladybird is also found on coniferous trees, spe- assess the favourability of Scots pine forest for the repro- cifically Scots pine, Pinus sylvestris L. (Roy et al., 2016; duction and overwintering of H. axyridis. The study is an Brown and Roy, 2018; Holecová et al., 2018), Austrian extension to the winter surveys of native ladybirds in the pine, Pinus nigra Arn. (Adriaens et al., 2008) and Nor- presence of H. axyridis from the same forest (Holecová way spruce, Picea abies (L.) Karsten (J. Kulfan, P. Zach, et al., 2018). unpublished data). Judging by the seasonal occurrence of H. axyridis on Harmonia axyridis has strong dispersal capabilities Scots pine in the invaded range (Pendleton and Pendle- (Osawa, 2000; Koch, 2003) which has been reflected by ton, 1997–2020; Brown and Roy, 2018; Holecová et al., the very fast spread of this species across Europe (Brown 2018), we expected the presence of the ladybird in Scots et al., 2008; Roy et al., 2016). In newly invaded areas pine forest all year round (prediction 1). As H. axyridis H. axyridis tended to use urban and agricultural landscapes prefers habitats within urban and agricultural landscapes rather than semi-natural landscapes (Brown et al., 2008; over semi-natural landscapes in the early years of inva- Grez et al., 2014). Soon after establishment, however, sion (Brown et al., 2008; Grez et al., 2014), we expected the ladybird diversified to many host plants and different low densities of the ladybird on pine trees (prediction 2). habitats (Adriaens et al., 2008; Roy et al., 2016) track- Considering the migratory behaviour of H. axyridis to and ing aphid populations (Honěk et al., 2019). Adults migrate from overwintering sites (Hodek et al., 1993; Jeffries et from breeding to overwintering sites (Obata, 1986; Roy al., 2013), we expected that this behaviour would be re- and Brown, 2015) and may form large aggregations (Pani- flected in the seasonal dynamics of the ladybird (prediction 3). gaj et al., 2014). The overwintering sites of H. axyridis are principally man-made structures in urban areas and, less often, outside these areas in landscapes with rocks, Materials and methods trees and forests providing shelter from the worst of the weather (Hodek et al., 2012; Panigaj et al., 2014; Nedvěd, Study area 2015). Understanding of habitat preference and habitat suit- Our study was carried out in the central part of the Borská ability has wide relevance to invasion biology of eurytopic nížina lowland within the forest complex Bor in south- species such as H. axyridis (Roy et al., 2016). Sequential west Slovakia, Central Europe. Monoculture stands of investigations of changes in the densities of H. axyridis Scots pine on sand dunes (Šomšák and Kubíček, 1994), as well as many other insects on trees in parks, forests replacing the former oak-pine forests (Pino-Quercetum; or other habitats are crucial in this context but there are Kollár et al., 2011), characterize the area. The climate is limitations to them in temperate regions. Leaf fall, for ex- sub-continental, with mean annual temperatures from 9.0 ample, affects the timing and duration of investigations of to 9.6°C and mean temperatures in January between –1.0 H. axyridis on deciduous trees and shrubs. Within Europe, and –2.0 °C (Bochníček et al., 2015). The two successive Brown and Roy (2018) studied the changes in the number summers of 2013 and 2014 were very warm and very dry of H. axyridis on mature lime trees Tilia × europea L. from (Holecová et al., 2016) and the winters of 2013/2014 and April to October, and Honěk et al. (2019) investigated the 2014/2015 were both exceptionally mild (Holecová et al., seasonal dynamics of H. axyridis on lime, maple (Acer 2018). spp.) and birch trees (Betula spp.) from late April to mid Mononoculture Scots pine stands aged 5 to 100 years November. In contrast to deciduous trees, evergreen con- were sampled. Four stands comprised closed canopy stands

82 We modelled seasonal changes in the abundance of H. axyridis using the Generalized

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Then, Then, Wethe in we we pooledabundancethe modelledmodelled abundance the datathe ofthe dataabundance H. fromabundance axyridis collected 10 ofsamples H. perofrepeatedly axyridisH. 200 withiaxyridis branches nperin the the200 stand samebranches as a for standsfunction eachas avia functionsampling ofrandom of autocorrelation in the abundance data collected occassion.standrepeatedly effect. Then,in Wethe we samepooled modelled stands the the datavia abundance randomfrom 10 ofsamples H. axyridis withi pern the200 standbranches for aseach a function sampling of Table 1. stand effect.occassion.per We 200 pooled branches Then, the we data asmodelled afrom function 10the samplesabundance of the factorswithi of H.n year,theaxyridis stand canopy per for 200 each branches sampling as a function of the factors year, canopyoccassion.standthe factor effect. closure sThen, year, We weand canopypooled modelled age closure theof thedatapine andabundance fromstand age of10 as ofpinesamples parametricH. standaxyridis withi as parametricper nfixed the200 standbranchesterms, fixed for the terms, aseach afactor function thesampling factor of stand effect. 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Then, we modelledstand the abundance (sample areaof H.standthethe effect)axyridis factorfactor (sample sas per standyear, a 200arearandom canopy (sample brancheseffect) teclosure asrm, areaas a randomaand functioneffect)and the age te seasonalrm,ofasof a pineandrandom thestand effect seasonal term, as (inparametric and effect month (in fixed granularity)month terms, granularity) the as factor as the factors year,stand canopy (sample closure area effect) and age as aof random pine stand term, as and parametric the seasonal fixed effect terms, (in themonth factor granularity) as Harmonia axyridis standthethe factorseasonalnon (sample-parametrics year, areaeffect canopy effect) smooth (in closureas monthterm a random thatandgranularity) wasage term, allowedof andpine as the tostandthe seasonalchange non-para as parametric between effect- (in the fixed month stands terms, granularity) with the open factor and as Sampling of the factors year, canopy closurethe nonand -ageparametric of pine thestandstand smooth non (sampleas-parametric parametric term area that effect)smooth fixed was as terms,allowedterm a random that the was tofactor te change rm,allowed and between theto change seasonal thebetween effect stands (inthe with monthstands open granularity)with andopen and as stand (samplethe area non effect)-parametric as a randomsmooth tetermrm, thatand wasthe seasonalallowed toeffect change (in monthbetween granularity) the stands aswith open and themetricstandclosed non (sample - canopyparametricsmooth area(i.e. term smootheffect)allowing that asterm was fora random thatstand*seasonal allowed was te allowedrm, to and change interaction). tothe change seasonal between between effect the(in monthstands withgranularity) open and as stand (sample area effect) as a random term, and theclosedthe seasonalnon canopy-parametric effect (i.e. (inallowingsmooth month term for granularity) stand*seasonalthat was allowedas interaction). to change between the stands with open and Over a two year period (April 2013–Marchclosed 2015) canopythe adultnon- (i.e.parametric closedthe allowing stands canopysmooth forwith (i.e.term stand*seasonal open allowing that andwas for allowedclosed stand*seasonal interaction). canopyto change (i.e. interaction).between allowing the stands for with open and closedthe non canopy-Theparametric semiparametric (i.e. allowing smooth term fornegative stand*seasonal that wasbinomial allowed GAMinteraction). to changemodel fobetweenr the abundance the stands of with H. axyridisopen and Y the non-parametric smooth term that was allowed closedto change canopyThe between semiparametric (i.e. allowingthe stands negative for with stand*seasonal open binomial and GAM interaction). model fo r the abundance of H. axyridis Y H. axyridis were collected in the south-eastern marginsTheclosed semiparametric ofcanopy stand*seasonal (i.e. allowingThe negativesemiparametric for interaction). stand*seasonal binomial negative GAM interaction). binomial model GAM for themodel abundance for the abundance of H. axyridis of H. axyridis Y Y closedin time Thecanopy t (indexed semiparametric (i.e. inallowing months negative forfrom stand*seasonal the binomial beginning GAM interaction). of the model study) fo r and the standabundance s was ofspecified: H. axyridis Y monoculture pineclosed stands. canopy Collections (i.e. allowing were for made stand*seasonal monthly, interaction).in timeThe Thet (indexed semiparametric semiparametric in months negative fromnegative the binomial beginning binomial GAM of GAMthe model study) model fo andr the stand abundance s was specified:of H. axyridis Y The semiparametricin time t (indexed negative in months binomial from GAMthe beginning model fo ofr the abundancestudy) and standof H. saxyridis was specified: Y in the middle of each month except for Novemberin time t (indexed 2013 infor timemonths the tThe (indexedabundance semiparametricfrom inthe months of beginning H. negativefromaxyridis the of binomial beginning theY in study) time GAM of andtthe (indexedmodel study) stand fo and r s inthe was stand abundance specified: s was specified: of H. axyridis Y The semiparametric negative binomial GAM in timemodel t (indexed for the abundance in months offrom H. axyridisthe beginning Y of the study) and stand s was specified: in time t (indexed in months from the beginning of the study) and stand s was specified: when logistical difficulties did not allow sampling. Har- monthsin time t (indexedfrom the in beginning months from of thethe beginning study) and of the stand study) s was and stand s was specified: in time t (indexed in months from the beginning of the study) and stand s was specified: ( ) monia axyridis was recorded on days with favourable specified: ~ , ~ ( , ) ~ ( , ) weather conditions (dry and not too windy) between 10:00 ( 𝑠𝑠𝑠𝑠 ) ( 𝑠𝑠𝑠𝑠 ) with ~ 𝑌𝑌𝑠𝑠𝑠𝑠~,𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝜇𝜇𝑠𝑠𝑠𝑠, 𝜃𝜃 and 14:00 h CET by tree beating. Each sample constitu- ~ 𝑌𝑌(𝑠𝑠𝑠𝑠 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁, ) 𝜇𝜇𝑠𝑠𝑠𝑠 𝜃𝜃 ~ (with, ) 𝑌𝑌𝑠𝑠𝑠𝑠 ~𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 (𝜇𝜇𝑠𝑠𝑠𝑠 ,𝜃𝜃 ) with 𝑠𝑠𝑠𝑠 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑠𝑠𝑠𝑠 ted specimens from 20 low (up to 3 m above the ground) with 𝑌𝑌 𝑠𝑠𝑠𝑠𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑌𝑌𝜇𝜇𝑠𝑠𝑠𝑠𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝜃𝜃 𝜇𝜇 𝜃𝜃 with with𝑠𝑠𝑠𝑠 𝑠𝑠𝑠𝑠 ( ) = + 𝑌𝑌. ( 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁= 𝑠𝑠𝑠𝑠𝜇𝜇) + 𝜃𝜃 . ( 𝑠𝑠𝑠𝑠 = ) + . ( = ) + branches of several close-growing trees beaten over𝑌𝑌 a𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 cir- with𝜇𝜇 (𝜃𝜃 ) = + . ( = 𝑌𝑌) + 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁. ( 𝜇𝜇 𝜃𝜃 = ) + . ( = ) + with ( ) ( ) ( ) ( ) cular canvas beating tray of 1.0 m diameter. A total of 10 ( 𝑠𝑠𝑠𝑠) = 0+ 𝑟𝑟. ( 𝑡𝑡= ) + 𝑐𝑐. ( 𝑠𝑠= ) + 𝑎𝑎. ( 𝑎𝑎= ) + 𝑠𝑠 𝑙𝑙𝑙𝑙𝑙𝑙( 𝜇𝜇𝑠𝑠𝑠𝑠) = 𝛽𝛽0 + ∑ 𝛽𝛽𝑟𝑟. 𝐼𝐼( 𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑡𝑡 = 𝑟𝑟) + ∑ 𝛾𝛾𝑐𝑐. 𝐼𝐼( 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑠𝑠 = 𝑐𝑐) + ∑ 𝛿𝛿𝑎𝑎. 𝐼𝐼( 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 = 𝑎𝑎) + 𝑙𝑙𝑠𝑠 samples were taken in each stand on every sampling( ) =date.( ) += 𝑙𝑙𝑙𝑙𝑙𝑙+𝜇𝜇𝑠𝑠𝑠𝑠. ( .𝛽𝛽(0 ∑=𝑟𝑟 =𝛽𝛽)𝑟𝑟 (+𝐼𝐼) +𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑡𝑡 ).. 𝑟𝑟(( ∑𝑐𝑐 𝛾𝛾𝑐𝑐 =𝐼𝐼=𝑐𝑐)𝑐𝑐𝑐𝑐) +𝑐𝑐𝑐𝑐) +𝑐𝑐𝑐𝑐𝑠𝑠 .𝑐𝑐( . (∑𝑎𝑎=𝛿𝛿𝑎𝑎 )𝐼𝐼=+𝑎𝑎𝑎𝑎𝑎𝑎)𝑎𝑎 + 𝑎𝑎 𝑙𝑙𝑠𝑠 𝑙𝑙𝑙𝑙𝑙𝑙 (𝜇𝜇𝑠𝑠𝑠𝑠 ) 𝛽𝛽0 +∑𝑟𝑟 𝛽𝛽𝑟𝑟 𝐼𝐼 (𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑡𝑡 . 𝑟𝑟 ) ∑𝑐𝑐 𝛾𝛾𝑐𝑐 =𝐼𝐼 (𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑠𝑠 𝑐𝑐 ) ∑𝑎𝑎 𝛿𝛿𝑎𝑎 𝐼𝐼 (𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎 ) 𝑙𝑙𝑠𝑠 ( ) = + . ( = ) + . (𝑙𝑙𝑙𝑙𝑙𝑙 𝜇𝜇𝑠𝑠𝑠𝑠 ==𝛽𝛽)0 ++∑ 𝛽𝛽𝑟𝑟(..𝐼𝐼(𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑡𝑡)=.=(𝑟𝑟) ++∑ 𝛾𝛾𝑐𝑐=.𝐼𝐼 𝑐𝑐)𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑠𝑠 =𝑐𝑐 +∑ 𝛿𝛿𝑎𝑎 .𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 =𝑎𝑎 +𝑙𝑙𝑠𝑠 In total 32,200 branches (20 branches × 10 samples × 7 + 𝑟𝑟 . 𝑐𝑐 = 𝑎𝑎 𝑠𝑠𝑠𝑠 𝑙𝑙𝑙𝑙0𝑙𝑙 𝜇𝜇 𝑟𝑟 𝛽𝛽 + ∑𝑡𝑡 𝛽𝛽 ( 𝐼𝐼 𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦 ).𝑐𝑐 (𝑟𝑟 ∑ 𝛾𝛾𝑠𝑠=𝐼𝐼 𝑐𝑐) 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑎𝑎 𝑐𝑐 ∑𝑎𝑎 𝛿𝛿 𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎𝑠𝑠 𝑎𝑎 𝑙𝑙 𝑠𝑠𝑠𝑠𝑙𝑙𝑙𝑙𝑙𝑙 𝜇𝜇 0 𝛽𝛽 ∑𝑟𝑟 𝛽𝛽 𝐼𝐼 𝑦𝑦𝑦𝑦𝑡𝑡 𝑦𝑦𝑦𝑦𝑟𝑟 𝑐𝑐𝑟𝑟 ∑𝑡𝑡𝑐𝑐𝛾𝛾 𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑠𝑠 𝑠𝑠 𝑐𝑐 ∑ 𝛿𝛿 𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎 𝑙𝑙 𝑠𝑠 𝑠𝑠𝑠𝑠 0 𝑟𝑟 𝑡𝑡 𝑐𝑐 𝑠𝑠𝑠𝑠𝑠𝑠 0+ ∑ 𝑠𝑠𝑐𝑐𝑎𝑎𝑟𝑟( 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ𝑎𝑎𝑡𝑡𝑡𝑡). 𝐼𝐼( 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑠𝑠 𝑐𝑐𝑐𝑐𝑠𝑠 =𝑐𝑐 𝑐𝑐) 𝑠𝑠 𝑎𝑎 𝑎𝑎 𝑠𝑠 stands × 23 months),𝑙𝑙𝑙𝑙𝑙𝑙 each𝜇𝜇 1 m𝛽𝛽 long,∑ were𝛽𝛽 𝐼𝐼 𝑦𝑦 sampled.𝑦𝑦𝑙𝑙𝑙𝑙𝑦𝑦𝑦𝑦𝑙𝑙 𝜇𝜇𝑟𝑟 Young∑𝛽𝛽𝛾𝛾 𝐼𝐼 ∑𝑐𝑐𝑙𝑙𝑙𝑙𝑐𝑐𝑐𝑐𝑙𝑙𝑐𝑐𝑐𝑐𝛽𝛽𝜇𝜇𝑐𝑐𝑐𝑐𝑟𝑟 𝐼𝐼 𝑦𝑦𝑦𝑦𝑐𝑐𝛽𝛽𝑦𝑦𝑦𝑦 ∑∑𝑐𝑐 𝑟𝑟𝛿𝛿𝛽𝛽𝑐𝑐 𝐼𝐼𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎∑𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑐𝑐 𝛾𝛾𝑡𝑡 𝐼𝐼𝑎𝑎𝑟𝑟𝑐𝑐𝑐𝑐𝑐𝑐𝑙𝑙𝑐𝑐𝑐𝑐∑𝑐𝑐𝑐𝑐𝑠𝑠𝛾𝛾 𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎 ∑ 𝛿𝛿𝑐𝑐 𝐼𝐼 ∑𝑎𝑎𝑎𝑎𝑎𝑎𝛿𝛿 𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑙𝑙𝑎𝑎 𝑙𝑙 𝑟𝑟 + ( ∑𝑟𝑟)𝑠𝑠. 𝑐𝑐(𝑚𝑚𝑚𝑚𝑚𝑚𝑐𝑐𝑚𝑚ℎ𝑡𝑡 =𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐) 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑠𝑠 𝑐𝑐 𝑎𝑎 𝑎𝑎 𝑟𝑟 𝑐𝑐 ∑𝑐𝑐𝑎𝑎 𝑠𝑠𝑐𝑐 (𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ𝑡𝑡 ) 𝐼𝐼 (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑠𝑠 𝑐𝑐 ) pines (5 years old) were sampled+ as whole( trees.). ( In each =+ ) ( +)∑. (𝑠𝑠𝑐𝑐 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ𝑡𝑡 =.𝐼𝐼 𝑐𝑐)𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐~𝑠𝑠 (=0,𝑐𝑐 ) ∑𝑐𝑐 𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ 𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐~𝑐𝑐𝑐𝑐 (0, 𝑐𝑐 ) 𝑐𝑐 𝑐𝑐𝑡𝑡 𝑠𝑠 ( 2) stand, H. axyridis were always obtained from different ∑ 𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ 𝐼𝐼𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑡𝑡 𝑐𝑐 𝑠𝑠~ 𝑠𝑠(0, ) 𝑐𝑐 𝑡𝑡 𝑠𝑠 𝑐𝑐 𝑐𝑐 𝑡𝑡 𝑠𝑠 2 ∑ 𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ 𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 ∑ 𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ 𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑙𝑙𝑠𝑠~𝑐𝑐𝑐𝑐𝑁𝑁(0, 𝑐𝑐𝜎𝜎2) trees which were marked to avoid repeated𝑐𝑐 sampling of the ∑where:𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ 𝑐𝑐𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 𝑙𝑙 𝑁𝑁 𝜎𝜎2 where: ~ (0, )𝑙𝑙 𝑠𝑠 𝑁𝑁 𝜎𝜎 ~ (0, where:𝑐𝑐) 𝑙𝑙 ~𝑁𝑁 (0,𝜎𝜎2 ) same trees. Collected beetles were placed in 70% ethanol where: ~ (0, 2)𝑙𝑙 𝑠𝑠 𝑁𝑁 𝜎𝜎 where:2 is the intercept, 𝑙𝑙𝑠𝑠 𝑁𝑁 𝜎𝜎 2 and counted later in the laboratory. The larvae of H. axyridiswhere:𝑠𝑠 where: is the intercept, 2 𝑙𝑙𝑠𝑠 𝑁𝑁 𝜎𝜎 where: 𝑙𝑙 𝑁𝑁 𝜎𝜎where: 𝑠𝑠 were recorded qualitatively (present/absent). 0 is the intercept, 𝑙𝑙 𝑁𝑁 𝜎𝜎 where: 𝛽𝛽0 isis the .the( intercept intercept,=, ) is the intercept𝛽𝛽 , is the saturated effect of year (with the usual identifiability constraint – 𝛽𝛽0 . ( = ) is the saturated effect of year (with the usual identifiability constraint – is the intercept, 𝛽𝛽0 is .the( intercept= , ) is the saturated effect of year (with the usual identifiability constraint – Data analysis 𝛽𝛽we𝑟𝑟 use 𝑟𝑟. (d “contr 𝑡𝑡 = ast )treatment isis thethe saturated saturated” or baseline effect effect constraints of yearof year(with for (withall the factors usual the inidentifiability the model), constraint – 0 we∑𝑟𝑟 use𝛽𝛽𝑟𝑟. d𝐼𝐼( “𝑦𝑦contr𝑦𝑦𝑦𝑦𝑦𝑦𝑡𝑡 ast= treatment𝑟𝑟) is the saturated” or baseline effect constraints of year (with for all the factors usual in identifiability the model), constraint – 0 is the𝛽𝛽 intercept,we∑ 0 used “contrast treatment” or baseline constraints for all factors in the model), . ( we∑usual𝛽𝛽𝑟𝑟= 𝛽𝛽use𝑟𝑟 𝐼𝐼)didentifiability is𝑦𝑦“contr𝑦𝑦 the𝑦𝑦𝑦𝑦𝑡𝑡 astsaturated 𝑟𝑟treatment constrainteffect” or of baseline year – (withwe constraints used the usual“contrast for identifiabilityall factors treat- in the constraint model), – 𝛽𝛽 . ( = ) is the saturated effect of year∑we (with 𝛽𝛽use.𝐼𝐼d the( 𝑦𝑦“contr𝑦𝑦 𝑦𝑦𝑦𝑦usualast= 𝑟𝑟identifiabilitytreatment) is the saturated” or constraintbaseline effect constraints – of year (withfor all the factors usual in identifiability the model), constraint – ∑wement”𝑟𝑟 𝛽𝛽use𝑟𝑟 .𝐼𝐼d or( 𝑦𝑦“contr𝑦𝑦 baseline𝑦𝑦𝑦𝑦𝑡𝑡 ast 𝑟𝑟treatment= constraints) is the” or forest baseline for stand all constraintsfactors closure ineffect forthe all, model), factors in the model), In order to gain insights into how frequently0 H. axyridis∑we𝑟𝑟 use𝑟𝑟 d “ contr𝑡𝑡 ast. treatment( ” or= baseline) is the constraints forest stand for closure all factors effect in, the model), we𝑟𝑟 use𝑟𝑟 d “contr𝑡𝑡 ast treatment”𝛽𝛽 or baseline. ( constraints𝛽𝛽 𝐼𝐼 𝑦𝑦=𝑦𝑦𝑦𝑦𝑦𝑦we) 𝑟𝑟for use𝑟𝑟 .𝑟𝑟all(d factors“contr𝑡𝑡 ast in =treatmentthe) model)” ,or baseline constraints for all factors in the model), occurred in Scots∑ pine𝛽𝛽 𝐼𝐼forest𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦 and𝑟𝑟 how likely it was to rec- ∑ 𝑐𝑐is𝛽𝛽 𝑐𝑐.the𝐼𝐼( 𝑦𝑦 𝑦𝑦saturated 𝑦𝑦𝑦𝑦 𝑠𝑠=𝑟𝑟 )effect isis thethe forestof forest year stand stand (with closure closure the effect usual effect,, identifiability constraint – ∑ 𝛾𝛾. 𝐼𝐼( 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 = 𝑐𝑐) is the forest stand closure effect, ( ∑𝑐𝑐 𝛾𝛾𝑐𝑐 𝐼𝐼. 𝑐𝑐(𝑐𝑐𝑐𝑐) 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐=𝑠𝑠 ) 𝑐𝑐is the saturated forest stand effect, ord this species in the surveyed stands throughout the year,. 𝑐𝑐 𝛾𝛾𝑐𝑐 .=𝐼𝐼 (𝑐𝑐𝑐𝑐𝑐𝑐 is𝑐𝑐𝑐𝑐 the𝑐𝑐𝑐𝑐=𝑠𝑠 forest) is𝑐𝑐 the stand saturated closure forest effect stand, effect, . ( = ) is the∑we forest𝑟𝑟 use𝑟𝑟 standd “contr closure𝑡𝑡 ast effect∑treatment𝛾𝛾 ,. 𝐼𝐼 (𝑐𝑐 𝑐𝑐𝑐𝑐 ”𝑐𝑐𝑐𝑐 or𝑐𝑐𝑐𝑐 baseline = ) is𝑐𝑐 is ) the is the thesaturatedconstraints forest saturated foreststand for closure foreststand all factorseffect effect stand, , in effect,the model) , 𝛽𝛽 𝐼𝐼 𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦 𝑟𝑟 𝑐𝑐𝑎𝑎 𝑐𝑐 𝑎𝑎. ( 𝑎𝑎=𝑠𝑠 ) is the saturated forest stand effect, we calculated the frequency of occurrence of H. axyridis𝑐𝑐 𝑐𝑐 ∑∑ 𝛾𝛾𝑠𝑠 𝐼𝐼 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐 ∑ 𝑎𝑎 𝛿𝛿𝑎𝑎. 𝐼𝐼( 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 = 𝑎𝑎) is the saturated forest stand effect, 𝑐𝑐 𝑐𝑐 𝑠𝑠 𝛾𝛾 .𝐼𝐼 (𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐∑ 𝑐𝑐𝑐𝑐𝑐𝑐 =is𝛿𝛿𝑐𝑐 the)𝐼𝐼the is𝑐𝑐 𝑎𝑎𝑎𝑎𝑎𝑎random the random saturated 𝑠𝑠(normally𝑎𝑎 (normally forest distributed) stand effectdistributed) sample, area sample effect, area as the percentage∑ of𝛾𝛾 the𝐼𝐼 𝑐𝑐 𝑐𝑐𝑐𝑐number𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 of𝑐𝑐 stands in which the ∑∑ 𝑎𝑎is𝛿𝛿𝛾𝛾 𝑎𝑎the.𝐼𝐼 (random𝑐𝑐𝑎𝑎𝑎𝑎𝑎𝑎𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎 𝑐𝑐𝑐𝑐= (normally𝑎𝑎 ) 𝑐𝑐 distributed) sample area effect, . ( = ) is the saturated. forest( stand effect=∑ 𝑎𝑎is𝛿𝛿, ) 𝑎𝑎the is𝐼𝐼 random𝑎𝑎𝑎𝑎𝑎𝑎the 𝑎𝑎forest (normally𝑎𝑎 standis the saturateddistributed)closure foresteffect sample stand, area effect effect, , ladybird was found during the observation period April ∑effect,𝑠𝑠 is𝛿𝛿 the𝐼𝐼 random𝑎𝑎𝑎𝑎𝑎𝑎 (normally𝑎𝑎 distributed) sample area effect, 𝑎𝑎 𝑎𝑎 𝑎𝑎𝑙𝑙𝑠𝑠 is the random (normally distributed) sample area effect, 𝑎𝑎 𝑎𝑎 𝑎𝑎 ∑ 𝛿𝛿 𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎 𝑎𝑎 𝑎𝑎 𝑐𝑐 𝑐𝑐 is the random𝑠𝑠 𝑙𝑙∑𝑠𝑠 ( 𝛿𝛿(normally 𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎 ) is distributed)is a a𝑎𝑎smooth smooth (penalizedsample (penalized area spline effect spline ,modelled) modelled) season seaal- effect which is allowed to differ 2013–March 2015∑ isin𝛿𝛿 the relation𝐼𝐼 random𝑎𝑎𝑎𝑎𝑎𝑎 to (normally𝑎𝑎 the total distributed) ∑number𝛾𝛾 𝐼𝐼 𝑐𝑐 ofsample𝑐𝑐𝑐𝑐 sur𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐- area𝑙𝑙 effect is𝑐𝑐 the, random (normally distributed) sample area effect, . ( = 𝑙𝑙sonal)𝑠𝑠 is the effect saturated which forest is allowed stand effectto differ, between open and veyed stands (n = 7). 𝑠𝑠 between𝑐𝑐 open𝑡𝑡 and closed canopy forest stands. Because seasonal effect is necessarily 𝑠𝑠 𝑙𝑙 𝑠𝑠𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚ℎ We modelled𝑙𝑙 seasonal changes in the abundance of closed𝑙𝑙 canopy forest stands. Because seasonal effect is ne- ∑𝑎𝑎 𝛿𝛿𝑎𝑎 𝐼𝐼 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎periodic, we enforced periodicity (and smoothness in the whole periodic function) by H. axyridis using the Generalized Additive Model is the (GAM)random (normallycessarily distributed)periodic, we sample enforced area periodicity effect, (and smooth- (Hastie and Tibshirani, 1990; Wood, 2017) with nega- nesspenalized in the cyclic whole spline periodic, function) by penalized cyclic 𝑠𝑠 tive binomial distribution (overdispersion𝑙𝑙 taken into ac- spline. ( ) = + . is a parameter related inversely to the overdispersion, namely 2 𝜇𝜇𝑠𝑠𝑠𝑠 𝜃𝜃 𝑉𝑉𝑉𝑉𝑉𝑉 𝑌𝑌𝑠𝑠𝑠𝑠 𝜇𝜇𝑠𝑠𝑠𝑠 𝜃𝜃 Table 1. Characteristics of Scots pine stands (n =7) (stand i.d., site, geographicalThe model coordinates, was fitted age simultaneouslyof pine trees, canopy via penalized closure, likelihood optimization, with habitat description). LNV, Lakšárska Nová Ves; STUD, Studienkacrossvalidated choice of penalization constants for the smooth terms (WOOD, 2008). Stand Site Longitude Latitude Age CanopyStatistical analyses were performedHabitat in R (R CORE TEAM, 2020). The package mgcv in R 1 LNV 17.1758 48.5822 60 Open(W OOD,Solitary 2017) was pine used trees to onapply sand the dunes, GAM no modelling. undergrowth 2 LNV 17.1761 48.5816 10 Closed Dense pine forest, no undergrowth 3 LNV 17.1727 48.5808 25 Closed Dense pine forest, no undergrowth 4 LNV 17.1644 48.5819 15 ClosedResults Dense pine forest, no undergrowth 5 STUD 17.1644 48.5402 100 Closed Dense pine forest, scattered undergrowth

6 STUD 17.1375 48.5377 20 Open Patches of pine trees, rich grassy undergrowth From April 2013 to March 2015 adult H. axyridis were recorded in each month of the year 7 STUD 17.1369 48.5416 5 Open Young pine trees in plantation, no undergrowth and in each pine stand (n = 7). In particular months, however, the ladybird was not recorded

in all stands (F < 100%). The ladybird occurred most frequently in April and May, and between October and December, and least frequently in February.83 The highest frequency of H. axyridis (F = 87%) indicated the presence of the ladybird in six of the seven stands, the lowest frequency (F = 14%) the presence in a single stand only (Fig. 1). Fig. 1 Fig. 1. Frequency of occurrence (%) of Harmonia axyridis in Scots pine forest throughout the year. The presence/absence data on the alien ladybird were collected monthly across pine stands (n = 7) in south-west Slovakia over April 2013–March 2015.

In total 500 adult H. axyridis were recorded, thus the numbers were low over the study period, within the range 0–195 specimens per 200 branches. Of the recorded beetles, 11% were found in spring (March–May), 5% in summer (June–August), 60% in autumn (September–November) and 24% in winter (December–February). The ladybirds were found most abundantly in November 2014 (195 specimens). During the winter of 2014/2015 H. axyridis abundance strongly decreased to 70 specimens in December, 41 specimens in January and just 7 specimens in February. The abundance of adult H. axyridis greatly varied between pine stands. The adults were found in highest numbers in the 15 year old closed canopy stand (stand 4, Table 1) which yielded 67% of the recorded specimens. They were least abundant in the patches of ( ) is a smooth (penalized spline modelled)( season)al is effect a smooth which (penalized is allowed spline modelled) to differ season al effect which is allowed to differ 𝑠𝑠between𝑐𝑐 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 ℎopen𝑡𝑡 and closed canopy forest stands𝑠𝑠between. 𝑐𝑐 Because𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 ℎopen𝑡𝑡 seasonaland closed effectcanopy isforest necessar stands.il Becausey seasonal effect is necessarily periodic, we enforced periodicity (and smoothnessperiodic, in wethe enforce wholed periodicityperiodic (andfunction) smoothness by in the whole periodic function) by penalized cyclic spline, penalized cyclic spline, H. (axyridis) = abundance+ . strongly decreased to 70 specimens is aa parameter parameter related related inversely inversely to the to overdispersion, the overdispersion, namely 2 namely in December, 41𝜇𝜇 𝑠𝑠𝑠𝑠specimens in January and just 7 speci- is a parameter related inversely to the overdispersion, namely ( ) = + . 𝑠𝑠𝑠𝑠 𝑠𝑠𝑠𝑠 𝜃𝜃 𝜃𝜃 2 𝑉𝑉𝑉𝑉𝑉𝑉mens𝑌𝑌 in February.𝜇𝜇 The model was fitted simultaneously𝜇𝜇𝑠𝑠𝑠𝑠 via penalized likelihood optimization, with The model was fitted𝑠𝑠𝑠𝑠 simultaneously𝑠𝑠𝑠𝑠 via penalized The abundance of adult H. axyridis greatly varied be- 𝜃𝜃 crossvalidated choice𝑉𝑉𝑉𝑉𝑉𝑉 𝑌𝑌of penalization𝜇𝜇 𝜃𝜃constants for the smooth terms (WOOD, 2008). The model was fitted simultaneously vialikelihood penalized optimization, likelihood with optimization, crossvalidated with choice of tween pine stands. The adults were found in highest num- Statisticalpenalization analyses constants were performedfor the smooth in R ( Rterms CORE (TWoodEAM, 2020)., bers The in thepackage 15 year mgcv old inclosed R canopy stand (stand 4, Table crossvalidated choice of penalization constants (2008).WforOOD ,theStatistical 2017) smooth was analysesused toterms apply were the (performedW GAMOOD modelling., 2008).in R (R Core 1) which yielded 67% of the recorded specimens. They Team, 2020). The package mgcv in R (Wood, 2017) was were least abundant in the patches of pine trees aged 20 Statistical analyses were performed in R (R CORE used T EAMto apply, 2020). the GAM The modelling. package mgcv in R years (stand 6) (2% of all specimens). The pattern of decrease in the number of H. axyridis from spring to summer, (WOOD, 2017) was used to apply the GAM modelling.Fig. 2. Seasonal variability of Harmonia axyridis counts. Plot of the ( ) component of the negative binomial Results followed by increase in the autumn and decrease during Resultsmodel (Scots pine stands with closed canopy). Solidthe line winter, – the was fitted recorded (predicted)𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 across abundancestands𝑡𝑡 1–5 but of Harmonianot stands axyridis, dotted 6–7 where low numbers𝑠𝑠 did𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 not ℎallow determination of From AprilApril 2013 2013 to to March March 2015 2015 adult adult H. axyridisH. axyridis werelines were recorded – theany 95%in seasonaleach confidence month trend. of the Inintervals. theyear 15 year old closed canopy stand andrecordedFig. in each 3. in Seasonal pineache stand month variab(n of= 7).theility In year particular of and Harmonia in months,each pine axyridis however, stand counts. theH. ladybird axyridis Plot was of was thenot not recorded recorded( in summer) component (June–August) of the n egative binomial Results (n = 7). In particular months, however, the ladybird was but it was recorded in high numbers in late autumn (No- in all stands (F < 100%). The ladybird occurred most frequently in April and May, and notmodel recorded (Scots in pineall stands stands (F with< 100%). open The canopy). ladybird Solid oc- linevember). – the fitted (predicted)𝑠𝑠𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 abundanceℎ𝑡𝑡 of Harmonia axyridis, dotted lines betweencurred most October frequently and December, in April and and least May, frequently and between in February. TheThe highest larvae frequencyof H. axyridis of were found in stands 1–6 in – the 95% confidence intervals. From April 2013 to March 2015 adult H. axyridisH.October were axyridis recordedand (F December, = 87%) in indicatedeach and leastmonth the frequentlypresence of the of in yearthe February. ladybird in lowsix ofnumbers, the seven in stands,most cases the as a single or few specimens, The highest frequency of H. axyridis (F = 87%) indicated from June to September. No larvae were recorded in the and in each pine stand (n = 7). In particular months,lowestthe however,presence frequency of thethe (F =ladybirdladybird 14%) the in presence wassix of not the in recordedsevena single stands, stand onlythe (Fig.5 year 1). old plantation (stand 7). In the 15 year old closed in all stands (F < 100%). The ladybird occurredFig.lowest most 1 frequency frequently (F = in14%) April the presence and May, in a singleand stand canopy stand (stand 4) the larvae of H. axyridis were not onlyFig. 1.(Fig. Frequency 1). of occurrence (%) of Harmonia axyridis in Scots pine forest throughoutfound the year.in summer The presence/absence (June–August). between October and December, and least frequentlydata onin the February. alien ladybird were The collected highest monthly acrossfrequency pine stands of(n = 7) in south-west SlovakiaThe negative over April 2013binomial–March GAM model showed that the 2015. factor year did not significantly affect the abundance of H. axyridis (F = 87%) indicated the presence of the ladybird in six of the seven stands, the H. axyridis (df = 2, χ2 = 0.837, p = 0.658). The abundance In total 500 adult H. axyridis were recorded, thus the numbers were low over the study lowest frequency (F = 14%) the presence in a single stand only (Fig. 1). of H. axyridis was significantly influenced by the age of period, within the range 0–195 specimens per 200 branches. Ofstand the recorded(df = 5, χbeetles,2 = 16.779, 11% p = 0.005), and it was signifi- Fig. 1 were found in spring (March–May), 5% in summer (Junecantly–August), higher 60% in thein 10,autumn 15 and 60 year old stands than in the 5 year old stand (baseline) (p < 0.05). The effect of month Fig. 1. Frequency of occurrence (%) of Harmonia axyridis in Scots(September pine forest– throughoutNovember) the and year. 24 %The in presence/absence winter (December –February).was The significant ladybirds wforere bothfound closed (edf = 5.611, 2χ = 42.74, data on the alien ladybird were collected monthly across pine stands (n = 7) in south-west Slovakia over April 2013–March 2 2015. most abundantly in November 2014 (195 specimens). During thep < 0.0001)winter of and 2014/2015 open canopy H. stands (edf = 4.396, χ = 12.97, p = 0.007). The effect of canopy closure proved not sig- axyridis abundance strongly decreased to 70 specimens in December, 41 specimens in nificant in influencing the number of H. axyridis (df = 1, In total 500 adult H. axyridis were recordedJanuary, thus theand justnumber 7 specimenss were in low February. over the study χ2 = 0.598, p = 0.439). The effect of forest stand (sample The abundance of adult H. axyridis greatly varied betweenarea effect)pine stands. was alsoThe not adults significant (edf = –2.803e-16,2 χ = 0.0, period, within the range 0–195 specimens per 200 branches. Of the recorded beetles, 11% p = 0.076). The model explained 61% of the deviance in were found in highest numbers in the 15 year old closed canopy stand (stand 4, Table 1) the abundance data with the overdispersion (θ = 0.686). were found in spring (March–May), 5% in summerwhich yielded (June 67%– August),of the recorded 60% specimens. in autumn They were least abundantThe seasonal in the patcheschanges of in the abundance of H. axyridis

(September–November) and 24% in winter (DecemberFrequency of Harmonia axyridis (%) –February). The ladybirds were found were shown in the GAM model separately for the stands with closed canopy (Fig. 2) and those with open canopy most abundantly in November 2014 (195 specimens). During the winter of 2014/2015 H. (Fig. 3). Bimodal seasonal variation was apparent in both cat- axyridis abundance strongly decreased to 70 specimens in December, 41 specimens in egories of stand, with peaks of H. axyridis in May and November. The seasonal abundance curves differed in January and just 7 specimens in February. Fig. 1. Frequency of occurrence (%) of Harmonia axyridis shape between the open and the closed canopy stands in in Scots pine forest throughout the year. The presence/ The abundance of adult H. axyridis greatlyabsence varied data between on the alien pine ladybird stands. were The collected adults monthly certain respects. The closed canopy stands hosted much Fig. 1 higher numbers of H. axyridis during the winter and were found in highest numbers in the 15 year oldacross closed pine canopystands (n =stand 7) in south-west(stand 4, Slovakia Table over1) April 2013–March 2015. showed a much greater decrease in the number of lady- which yielded 67% of the recorded specimens. They were least abundant in the patches of birds in the summer, in comparison to the open canopy stands. Judging from the overlapping confidence intervals, the size of the first abundance peak of H. axyridis did not In total 500 adult H. axyridis were recorded, thus differ significantly between the closed and open canopy the numbers3 were low over the study period, within the stands (Figs 2, 3). range 0–195 specimens per 200 branches. Of the recorded beetles, 11% were found in spring (March–May), 5% in summer2 (June–August), 60% in autumn (September– Discussion November) and 24% in winter (December–February). The ladybirds were found most abundantly in November Analysing systematically collected data, information

2014 1 (195 specimens). During the winter of 2014/2015 about the occurrence of H. axyridis in Scots pine forest

84 0 -1 Seasonal effect for closed stands for closed effect Seasonal -2 -3

2 4 6 8 10 12 Month Fig. 2 Fig. 2. Seasonal variability of Harmonia axyridis counts. Plot of the sclosed(montht) component of the negative binomial model (Scots pine stands with closed canopy). Solid line – the fitted (predicted) abundance of Harmonia axyridis, dotted lines – the 95% confidence intervals.

Fig. 3. Seasonal variability of Harmonia axyridis counts. Plot of the sopen(montht) component of the negative binomial model (Scots pine stands with open canopy). Solid line – the fitted (predicted) abundance ofHarmonia axyridis, dotted lines – the 95% confidence intervals. throughout the year has been gained, with the model of year round (prediction 1). However, we found relatively year-round changes in European populations of this lady- few adults and larvae in relation to sampling effort. Low bird presented for the first time. densities of H. axyridis may confirm that in the early years Habitat preferences in generalist species of ladybirds of invasion the ladybird focuses on habitats within urban are harder to interpret than in species with highly specified and agricultural landscapes rather than semi-natural land- ecologies (Majerus, 2016). Harmonia axyridis is adapted scapes (cf. Brown et al., 2008; Grez et al., 2014) (predic- to an extreme continental climate (Roy et al., 2016). We tion 2). recorded this species on Scots pine trees growing in poor The recorded changes in the number of H. axyridis soils (sandy dunes) with extreme climatic conditions all may reflect variation in habitats, microclimates and diet-

85 ary resources of the ladybird from one season of the year for foraging during the growing season and certain stands to another as well as its migratory behaviour. The bimodal for refuge during the winter. Many species of ladybirds pattern of seasonal dynamics of adult H. axyridis in Scots spend periods in dormancy in habitats that differ from pine forest, common for closed and open canopy stands, those uti-lized for reproduction and development (Hodek, involved two peaks reflecting the movement of H. axy- 2012; Ceryngier, 2015). ridis from and to overwintering sites (prediction 3). In the Dietary requirements obviously influence where a spe- spring, between March and April, deciduous trees lack cies lives and breeds (Majerus, 2016). Much greater de- foliage, and herbaceous plants are not yet available to the crease in the number of H. axyridis in the closed canopy ladybird: there is a lack of aphid prey on the plants. Thus stands than the open canopy stands in the summer could at this time evergreen Scots pine trees may intercept adult be explained by the ladybirdʼs seasonal preference for H. axyridis from various overwintering sites („brushing open habitats with more light and, possibly, higher prey effect of conifers“), resulting in the first abundance peak (aphids) availability (Honěk et al., 2018). Harmonia axy- of the ladybird in May, followed by decrease in abundance ridis appears to have a high ability to track aphid popula- during summer. The second (larger) peak in November re- tions in space and time (Osawa, 2000; Koch, 2003). In flects the migration of H. axyridis to overwintering sites. both the native and invaded ranges, seasonal changes in In temperate regions, late summer and early autumn is the number of H. axyridis are significantly correlated with a crucial time for ladybirds to build up fat reserves and those of aphids (Osawa, 2000; Honěk et al., 2019). We prepare for the winter (Majerus, 2016). Within Europe, found patchy colonies of a widely spread Scots pine aphid adult H. axyridis migrate to overwintering sites in October Cinara pini (L.) but did not record the number of this or (Roy and Brown, 2015; Panigaj et al., 2014). From mid other aphid species on pines. The Scots pine aphids are so October to mid November we found them frequently and mobile that the hatchlings of H. axyridis have difficulty abundantly on pine branches as a result of this characteris- capturing them in contrast to most arboreal aphids which tic migratory behaviour. We showed that the number of H. walk very slowly (Noriyuki and Osawa, 2016; Noriyuki, axyridis lowered markedly between mid November and 2011). The within-crown distribution of C. pini changes mid December and remained low during the rest of the over the season (Larsson, 1985) which may affect the dis- mild winter. The recorded low abundance of H. axyridis tribution of predators of this aphid on pines. on pines during the winter paralleled the trends in Britain High ability of prey searching and reproduction main- (Roy and Brown, 2015) where the ladybird is extreme- tains a stable population of H. axyridis in heterogen- ly difficult to find openly on Scots pine during this most eous and temporary habitats in the native range (Osawa, adverse time of the year for ladybirds (Pendleton and 2000). Within this range the habitats of H. axyridis can Pendleton, 1997–2020). be categorized as (1) habitats for survival and reproduc- Many ladybirds prefer sheltered sites such as crevices tion and (2) habitats for temporary refuge (Osawa, 2000), in rocks and in the bark of trees, buildings and monuments and the same habitat categorization may apply to the infor overwintering (Majerus, 1994; Koch and Galvan, vaded range of the ladybird. Our results showed that adult 2008; Roy and Brown, 2015), and the overwintering site H. axyridis were found in habitats within both categories, can be critical for their survival (Raak-van den Berg but with a prevalence for the second category. A question et al., 2012; Majerus, 2016). Harmonia axyridis within arises as to whether the Scots pine forest on sand dunes Europe usually overwinter in buildings (Roy and Brown, in Central Europe is a favourable habitat for H. axyridis. 2015) but they may also spend the winter in semi-natur- A favourable habitat was defined as one that allowed al habitats (Panigaj et al., 2014). Two of the three pine a species to both survive and breed (Southwood, 1977; stands with the highest abundance of H. axyridis (stand Majerus, 2016). Judging from the records of both adults 3 and 4) were closed canopy stands. One of them, the and larvae of H. axyridis on the lower branches of pine 15 year old stand (stand 4), yielded no H. axyridis dur- trees in most stands during the growing season, the Scots ing the two successive summers (June–August) but at- pine forest may be considered a favourable habitat for the tracted relatively high numbers of adult H. axyridis in late ladybird. However, as the lower branches of pines hosted autumn. Why did the beetles choose this particular stand few H. axyridis over the growing season, this microhabi- for overwintering? It is known that prominent landscape tat is regarded as inferior for the ladybird as a breeding features such as hills attract H. axyridis during the autumn habitat. The decreasing number of H. axyridis from spring migration (Hodek et al., 1993). Our study, however, was to summer, with very low numbers of the ladybird in pine conducted in a lowland area without any prominent land- stands in summer, did not parallel the seasonal dynam- scape features. Therefore, the local conditions providing ics of this species (increase in number during the growing protection of adults against wind, rain or snow, and par- season) on lime and maple trees in urban areas in Slovakia ticularly the stand characteristics such as the southern as- and Czech Republic (Viglášová et al., 2017). In agree- pect of forest edge, high tree density in the forest margin ment with this, H. axyridis rapidly dominated at lime tree (branches of closely-growing pines touching), and high sites in England but comprised a relatively small propor- degree of shelter offered by nearby old mature pine trees, tion of the ladybird assemblage at the Scots pine tree site could be crucial for H. axyridis selecting this particular (Brown and Roy, 2018). Regarding the weak persistence stand as an overwintering site. Our results suggest that of H. axyridis on the lower branches of pine trees during H. axyridis may alternate habitats in space and time; the the winter, choosing this microhabitat by the ladybird for ladybird apparently utilized certain pine stands preferably overwintering was suboptimal.

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