Release and post-release monitoring of the biocontrol agent “Trichilogaster acaciaelongifoliae” for the control of the invasive “Acacia longifolia” in Portugal - FOURTH REPORT

Responsáveis pela equipa:
Elizabete Marchante, emarchante@uc.pt
Hélia Marchante, hmarchante@esac.pt
20-12-2019
Centro de Ecologia Funcional da Universidade de Coimbra
Escola Superior Agrária do Instituto Politécnico de Coimbra
Release and post-release monitoring of the biocontrol
agent “Trichilogaster acaciaelongifoliae” for the
control of the invasive “Acacia longifolia” in Portugal
FOURTH REPORT

Release and post-release monitoring of the biocontrol agent “Trichilogaster
acaciaelongifoliae” for the control of the invasive “Acacia longifolia” in Portugal
Index
1. Preamble ............................................................................................................................................. 2
1. Release campaign in 2019................................................................................................................... 3
2. Post-release monitoring of the galls ................................................................................................... 5
3. Conclusions........................................................................................................................................ 12
4. References......................................................................................................................................... 13
Annex 1. FIRST REPORT – 2016.................................................................................................................. 14
Annex 2. SECOND REPORT – 2017............................................................................................................. 23
Annex 3. THIRD REPORT – 2018 ................................................................................................................ 32
Annex 4. FULL NAMES OF THE SITES REFERRED IN THE TEXT, FIGURES AND TABLES (2pages)................ 47

!
1. Preamble
This is the fourth annual report (previous reports available in Annex 1 to 3) about the release and post-
release monitoring of the biocontrol agent Trichilogaster acaciaelongifoliae for the control of the invasive
plant Acacia longifolia in Portugal.
Acacia longifolia is one of the most widespread invasive plants in coastal areas in Portugal. It causes
significant adverse ecological impacts (López-Núñez et al., 2017; Marchante et al., 2008, 2015; Figure 1,
bottom images), which may persist over time and make restoration of invaded areas increasingly more
difficult and complex (Le Maitre et al., 2011; Marchante et al., 2009, 2011a). In addition, it reduces forest
productivity, mainly in littoral pine plantations, and increases fire hazard (Le Maitre et al., 2011), with
consequent negative economic impacts. Control methods in use until recently are very expensive and
frequently unsuccessful, due to the persistent, extensive and long-lived seed-bank accumulated in the soil
(Marchante et al., 2010), that promotes the quick reinvasion of areas previously cleared.
Figure 1. Examples of coastal areas with native vegetation (top images) and areas densely invaded by Acacia longifolia, where the
invasive species is replacing the diverse native communities composed by several shrubs and herbaceous species by a dense
woody stand, almost monospecific (bottom images).
After more than 12 years of host speciﬁcity testing, risk assessment and permits (Marchante et al., 2011b;
Shaw et al., 2016), in November 2015, the biocontrol agent Trichilogaster acaciaelongifoliae (Australian bud-
galling wasp) started to be released in pre-selected sites along the Portuguese coast (Marchante et al., 2017,
Annex 1). This biocontrol agent is a highly host-specific organism, affecting almost exclusively A. longifolia
and targets mainly seed reduction; it is a small Australian gall-former (Hymenoptera: Pteromalidae, 3 mm),
with an annual life cycle (in average), spending most of the year inside the galls; emerging females search for

+
flower (and later vegetative) buds, oviposit the eggs, and die after 2 – 3 days (in average). As a result galls
are produced instead of flowers and as such seed production is prevented. In the short-term it reduces the
production and dispersal of seeds and in the longer-term a decrease of germination is expected after
control, fire or other disturbances, since the soil seed bank receives less and less seeds each year;
physiological stress to the plants may also be expected as they cannot cope with heavy gall loads (Dennill,
1985). This agent has been successfully used in South Africa for more than 30 years, where it decreased seed
production and vegetative growth (Dennill and Donnelly, 1991; Wilson et al., 2011). In Portugal, in Spring-
Summer 2016, 661
galls were detected in five2
of the release sites (Annex 1; Marchante et al., 2017), in 2017
around 1100 galls were detected in the same five sites (Annex 2)3
, and during 2018 the monitoring effort
detected almost 25000 galls in five sites (but while four sites were the same as in previous years, one was
new; one of the sites with establishment in 2016 burned in October 2017 and the population was lost, Annex
2) showing that establishment of T. acaciaelongifoliae is occurring in several sites.
1. Release campaign in 2019
As in previous campaigns, release and monitoring campaigns were done by a team from Centre for
Functional Ecology, which includes members of Escola Superior Agrária, Instituto Politécnico de Coimbra
(ESAC) and Universidade de Coimbra (UC). This work occurred in the context of two research project
(INVADER-IV- PTDC/AAGREC/4896/2014, funded by the Portuguese Foundation for Science and Technology,
and GANHA - POSEUR-03-2215-FC-000052, funded by Portugal 2020 through Programa Operacional
Sustentabilidade e Eficiência no Uso de Recursos, POSEUR).
Considering the establishment results of the 2018 release campaign (see below), in 2019 the releases of
Trichilogaster acaciaelongifoliae were limited to one site, at Vila Nova de Mil Fontes (Parque Natural
Sudoeste Alentejano e Costa Vicentina; Table I), since in the previous year it was not possible to release the
agent there; most of the other sites had establishment and therefore no further releases were done. Table I
includes all the release campaigns.
Table I. Number of female wasps of Trichilogaster acaciaelongifoliae released in the 5 release campaigns (2015-2019) in Portugal.
CODE SITE
#
COORDINATES PROJECT YEAR WASPS OBJECTIVE
$
ESP Esposende 41.508999,-8.784351 INVADER-B 2015 30 Biocontrol
SJD Dunas de São Jacinto 40.672717,-8.740729 INVADER-B 2015 88 Monitoring
EST Coimbra (ESAC) 40.216722,-8.450210 INVADER-B 2015 9 Founder pop.
TOC Tocha 40.34837,-8.81704 INVADER-B 2015 105 Monitoring
QUD Dunas de Quiaios 40.22476,-8.88622 INVADER-B 2015 80 Monitoring
SBV Serra da Boa Viagem 40.20037,-8.88969 INVADER-B 2015 65 Biocontrol
COI Coimbra (Pólo II) 40.18588,-8.41358 INVADER-B 2015 44 Monitoring
(Table continues next page)
1
In the first report, 56 galls were mistakenly reported due to an error in the summing of galls (all galls were correctly
identified in each site of release - Table 1, Annex 1 - but the sum formula had an error).
2
In the first report (Annex 1), galls were reported in four sites, but later on one gall was detected in Reserva Natural das
Dunas de São Jacinto, raising the number of sites with biocontrol agent resulting from the first release campaign to five.
3
In the second report (Annex 2), galls were reported in five sites, but later on a few galls were detected in Coimbra, in a
new site (Patos), raising the number of sites with T. acaciaelongifoliae to six. However, one site burned in October 2017
(Perímetro Florestal das Dunas de Cantanhede (Tocha)) and as such the total number of sites with establishment is five.

:
Table I (cont). Number of female wasps of Trichilogaster acaciaelongifoliae released during the five release campaigns in Portugal.
CODE SITE
#
$
COI1 Coimbra (Patos) 40.209237,-8.401237 INVADER-B 2015 39 Biocontrol
SPM São Pedro de Moel 39.75711,-9.02338 INVADER-B 2015 74 Monitoring
TOTAL 2015* 430
ESP Esposende 41.508999,-8.784351 INVADER-IV 2016 139 Biocontrol
SPI Espinho 40.923320,-8.658262 INVADER-IV 2016 135 Biocontrol
SJD Dunas de São Jacinto 40.672717,-8.740729 INVADER-IV 2016 74 Monitoring
EIX Eixo 40.620281,-8.568342 INVADER-IV 2016 54 Biocontrol
MIR Mira 40.394569,-8.789050 INVADER-IV 2016 45 Biocontrol
TOC1 Tocha N 40.320083,-8.845127 INVADER-IV 2016 129 Biocontrol
LAG Lagoa da Vela 40.269481,-8.799287 INVADER-IV 2016 70 Biocontrol
QUP Quiaios N 40.241645,-8.854573 INVADER-IV 2016 75 Biocontrol
EST Coimbra (ESAC) 40.216722,-8.450210 INVADER-IV 2016 83 Founder pop.
COI1 Coimbra (Patos) 40.209237,-8.401237 INVADER-IV 2016 38 Biocontrol
COV Covões 40.194632,-8.466302 INVADER-IV 2016 37 Biocontrol
ANO Anobra 40.161626,-8.510096 INVADER-IV 2016 21 Biocontrol
HEL Condeixa 40.113693,-8.513775 INVADER-IV 2016 5 Biocontrol
LAV Lavos 40.097376,-8.856496 INVADER-IV 2016 66 Biocontrol
LEI Leirosa 40.076190,-8.865008 INVADER-IV 2016 77 Biocontrol
MUR Mata do Urso 39.983285,-8.914007 INVADER-IV 2016 89 Biocontrol
PED Pedrogão 39.937940,-8.927712 INVADER-IV 2016 45 Biocontrol
VLE Vieira de Leiria 39.865863,-8.971235 INVADER-IV 2016 76 Biocontrol
PAT Paredes da Vitoria (Pataias) 39.707963,-9.048827 INVADER-IV 2016 138 Biocontrol
TOTAL 2016** 1396
ESP Esposende 41.508999,-8.784351 INVADER-IV 2017 39 Biocontrol
SEI Seixo 40.497200, -8.754436 INVADER-IV 2017 46 Biocontrol
EST Coimbra (ESAC) 40.216722,-8.450210 INVADER-IV 2017 32 Founder pop.
COV Covões
##
40.194632,-8.466302 INVADER-IV 2017 69 Biocontrol
ALH Alhadas (Cabecinho) 40.174834,-8.787518 INVADER-IV 2017 6 Biocontrol
FIG2 Figueira da Foz (McDonald´s) 40.166223,-8.853586 INVADER-IV 2017 6 Biocontrol
ANO Anobra 40.161626,-8.510096 INVADER-IV 2017 6 Biocontrol
ANO1 Anobra1 40.160576,-8.498345 INVADER-IV 2017 20 Biocontrol
FIG1 Figueira da Foz (Rotunda LIDL) 40.157502,-8.849582 INVADER-IV 2017 22 Biocontrol
VIV Vila Verde 40.154741,-8.795803 INVADER-IV 2017 16 Biocontrol
FIG Figueira da Foz 40.148682,-8.836959 INVADER-IV 2017 48 Biocontrol
MOR Morraceira 40.139481,-8.844876 INVADER-IV 2017 8 Biocontrol
GAL Gala, Parque de Merendas 40.123295,-8.859909 INVADER-IV 2017 10 Biocontrol
HEL Condeixa 40.113693,-8.513775 INVADER-IV 2017 2 Biocontrol
PAT Paredes da Vitoria (Pataias) 39.707963,-9.048827 INVADER-IV 2017 21 Biocontrol
LSA Lagoas de Santo André 37.991658,-8.854934 INVADER-IV 2017 46 Biocontrol
FAR Faro 37.028391,-8.005642 INVADER-IV 2017 28 Biocontrol
TOTAL 2017*** 425
RBA Riba de Âncora 41.808889,-8.796121 OTHERS 2018 48 Biocontrol
PEN Quinta Pentieiros 41.775557,-8.657753 GANHA 2018 25 Biocontrol
ESP Esposende 41.508999,-8.784351 GANHA 2018 83 Biocontrol
TOC4 Charco Berlengas 40.32656634,-8.7821874 GANHA 2018 4 Biocontrol
TOC3 Cruzamento Tocha 40.3254768,-8.8131098 GANHA 2018 9 Biocontrol

A
Table I (cont). Number of female wasps of Trichilogaster acaciaelongifoliae released during the five release campaigns in Portugal.
CODE SITE
#
$
ANH Anha 41.674618,-8.801127 GANHA 2018 9 Biocontrol
BEZ Barrinha de Esmoriz 40.963981,-8.647378 GANHA 2018 45 Biocontrol
EIX2 Eixo (Quinta de S. Francisco) 40.616127,-8.567075 OTHERS 2018 18 Biocontrol
IP3 Acesso IP3
##
40.586589,-8.015036 OTHERS 2018 3 Biocontrol
VAG Dunas Vagos (GANHA) 40.535932,-8.74104 GANHA 2018 146 Biocontrol
BZM Belazaima do Chão
##
POC1 Pocariça (Cantanhede1) 40.382712,-8.574368 OTHERS 2018 39 Biocontrol
COV Covões 40.194632,-8.466302 INVADER-IV 2018 2 Biocontrol
FIG2 Figueira da Foz (McDonald´s) 40.166223,-8.853586 INVADER-IV 2018 8 Biocontrol
FIG1 Figueira da Foz (Rotunda LIDL)
##
40.157502,-8.849582 INVADER-IV 2018 16 Biocontrol
FIG Figueira da Foz 40.148682,-8.836959 INVADER-IV 2018 12 Biocontrol
SOU Soure 40.099786,-8.619687 OTHERS 2018 45 Biocontrol
PAT Paredes da Vitoria (Pataias) 39.707963,-9.048827 GANHA 2018 77 Biocontrol
STC Praia do navio, Santa Cruz 39.144362,-9.371916 GANHA 2018 13 Biocontrol
CAR Carapinheira, Mafra 38.935492,-9.276104 OTHERS 2018 34 Biocontrol
CAP Praias da Costa da Caparica 38.599935,-9.20743 GANHA 2018 48 Biocontrol
ARR Estrada Setúbal - praias 38.496597,-8.930805 GANHA 2018 12 Biocontrol
SAD Estrada Tróia - Comporta 38.426573,-8.824844 GANHA 2018 46 Biocontrol
BRJ Brejinhos 38.031267, -8.808383 GANHA 2018 43 Biocontrol
SIN2 Monte Feio - Sines 2 37.996261, -8.842971 GANHA 2018 20 Biocontrol
LSA Lagoas de Santo André 37.991658,-8.854934 GANHA 2018 103 Biocontrol
SIN1 Monte Feio - Sines 1 37.981616, -8.845499 GANHA 2018 16 Biocontrol
FAR Faro 37.028391,-8.005642 GANHA 2018 109 Biocontrol
ANT Antas 41.61092,-8.80828 GANHA 2018 54 Biocontrol
VNR Vila Nova da Rainha
##
POC2 Pocariça (Cantanhede2) 40.37305,-8.579074 OTHERS 2018 21 Biocontrol
PQS Pesqueiro Sancha 38.02586,-8.82275 GANHA 2018 52 Biocontrol
TOC2 Tocha (Caniceira) 40.3476,-8.77368 GANHA 2018 79 Biocontrol
TOTAL 2018**** 1262
VNMF Vila Nova de Mil Fontes 37.714934, -8.7845 GANHA 2019 1 Biocontrol
TOTAL 2019***** 1
#
for simplification, names of sites were shortened, for full name please check Annex 4;
##
many trees were cut after
releases;
$
"Monitoring"- site being monitored in detail for establishment and effects of the BCA; "Biocontrol"- site
being monitored only for establishment of the BCA; "Founder pop."- “founder population” initially maintained in a
greenhouse - these labels changed along time, with the present info relating to 2019; *releases from 20/11 to 07/
12/2015, with South African galls; **releases from 12/11 to 13/12/2016, with South African galls; ***releases from
06/12/2017 to 08/01/2018, with South African galls; ****releases from 14/06 to 20/07/2018, with Portuguese galls;
***** release at 28/06/2019, with Portuguese galls; besides 1 wasp, 23 galls were left in the field and as such more
wasps were expected to emerge (female and males).
2. Post-release monitoring of the galls
From January to October 2019 all release sites were monitored. Depending on the size of the galls and their
detectability, for some sites more than one monitoring was performed in 2019; when this happened, the
maximum number of galls recorded was used for Table II. Because the number of sites to be monitored is

C
currently too high to screen them all in detail, a selection had to be made: some sites were selected to
detailed monitoring (sites signalled as “Monitoring” in Table I), while others were monitored with a less
demanding protocol whose only aim was to detect establishment of T. acaciaelongifoliae, recording
presence or absence of galls in the plants where galls were released (sites signalled as “Biocontrol” in Table
I). During 2019 monitoring it was no longer possible to count the very high number of galls in sites with
establishment in the previous years and as such (sub)Total in Table II corresponds to the sum of galls
counted only in sites with new establishment (2019), from wasps released in 2018, from Portuguese
populations. It is noteworthy that while in the previous years the number of first generation galls/site was
always low, at most in the order of magnitude in the tens, in 2019 in several sites this number was much
higher, sometimes several hundred, e.g., at Lagoas de Santo André or Dunas Vagos (GANHA) (Table II). This
difference is most probably because the wasps released in 2015 (establishment in 2016) were of South
African origin (life cycle was not synchronized with the season and phenology of A. longifolia in Portugal),
while in 2018 the releases were done for the first time with wasps originating from Portuguese populations,
and hence more synchronized not only with the phenology of the host-plant (A. longifolia), but also with
more adequate meteorological conditions. Resuming, in 2019, there is establishment along most of the
Portuguese Coast and some more inland sites, from Riba de Âncora, in the north, to Faro, in the south of the
country, in a total of 32 sites (Figure 2), most of them being the first year of establishment of T.
acaciaelongifoliae: almost 6500 galls of first generation (with few exception, please check notes in Table II)
were detected in 27 sites (Table II). In most of the six sites where there was no establishment in 2019, either
trees were cut or the number of A. longifolia plants was too low (please check notes in Table II). As Table I,
Figure 2 and Table II summarize the information not only of the fifth release and fourth monitoring
campaigns (in 2019), but include also the previous campaigns in order to give a complete picture.
Figure 2. Field sites where the biocontrol agent Trichilogaster acaciaelongifoliae was released along the Portuguese coast during
the first (2015, black dots in the legend), second (2016, dark red dots), third (2017, green dots), fourth (2018, blue dots) and fifth
(2019, pink dots) release campaigns. Sites where galls were detected (establishment of the biocontrol agent) are marked with
symbols in full, and sites with no establishment are open; for 2019 releases information about establishment it is not yet possible.
Source: (López-Núñez et al., n.d.)

G
As in previous years, in 2019 some galls were detected, including insects emerging, outside the expected
time, i.e., with the life cycle not synchronized with the majority of the other galls. E.g., in the last months of
2018 and of 2019 immature galls were detected in Pólo II from University of Coimbra and several other sites,
but in low numbers and too small to monitor in detail. Although it was again not possible to be certain of the
number of generations per year, since T. acaciaelongifoliae is still synchronizing its life cycle with the
Northern hemisphere, these galls resulted from wasps released in the 2015 campaign and were possibly the
fourth generation (alternatively, they could be late third or early fifth generation galls).
Table II. Number of galls of Trichilogaster acaciaelongifoliae detected during all post-release monitoring campaigns, including the
one in 2019, to which this reports refers specifically. Except for 2019, only sites and years where galls were detected are shown.
CODE SITE
MONITORING
YEAR
Nº of DETECTED
GALLS (max
$
)
COORDINATES
SJD Reserva Natural das Dunas de São Jacinto 2016 1 4067272,-874073
TOC Perímetro Florestal das Dunas de Cantanhede (Tocha) 2016 38 40.34837,-8.81704
TOC Perímetro Florestal das Dunas de Cantanhede (Tocha) 2017 29 40.34837,-8.81704
TOC Perímetro Florestal das Dunas de Cantanhede (Tocha)
#
2018 0 40.34837,-8.81704
QUD Mata Nacional das Dunas de Quiaios (Dunas Quiaios) 2016* 9 40.22476,-8.88622
QUD Mata Nacional das Dunas de Quiaios (Dunas Quiaios) 2017 73 40.22476,-8.88622
QUD Mata Nacional das Dunas de Quiaios (Dunas Quiaios) 2018 1039 40.22476,-8.88622
COI Coimbra (Pólo II from University of Coimbra) 2016 9 40.18588,-8.41358
COI Coimbra (Pólo II from University of Coimbra) 2017** 413 40.18588,-8.41358
COI Coimbra (Pólo II from University of Coimbra) 2018 5899 40.18588,-8.41358
COI1 Coimbra (Patos) 2016 0 4020924,-840124
COI1 Coimbra (Patos) 2017*** 21 4020924,-840124
COI1 Coimbra (Patos) 2018 123 4020924,-840124
SPM Mata Nacional de Leiria (São Pedro de Moel) 2016 9 39.75711,-9.02338
SPM Mata Nacional de Leiria (São Pedro de Moel) 2017 437 39.75711,-9.02338
SPM Mata Nacional de Leiria (São Pedro de Moel) 2018**** 16415 39.75711,-9.02338
RBA Riba de Âncora (Baldios de Riba de Âncora) 2019 153 41.80889,-8.79612
PEN Quinta Pentieiros 2019 26 41.77556,-8.65775
ANH Anha 2019 0 41.67462,-8.80113
ANT Antas 2019 51 41.61092,-8.80828
ESP Esposende
##
2019 2546 41.50899,-8.78435
BEZ Barrinha de Esmoriz 2019 77 40.96398,-8.64738
EIX2 Eixo (Quinta de S. Francisco) 2019 107 40.61613,-8.56707
IP3 Acesso IP3
###
2019 0 40.58659,-8.01504
VAG Dunas Vagos (GANHA) 2019 733 40.53593,-8.74104
BZM Belazaima do Chão
###
2019 0 40.52769,-8.33015
VNR Vila Nova da Rainha
###
2019 0 40.47055,-8.09458
POC1 Pocariça, Sr. Mário Mendes (Cantanhede) 2019 37 40.38271,-8.57437
POC2 Pocariça, vizinho Sr. Mário Mendes (Cantanhede) 2019 39 40.37305,-8.57907
TOC2 Tocha (Caniceira) 2019 11 40.34760,-8.77368
TOC4 Charco Berlengas 2019 3 40.32657,-8.78219
TOC3 Cruzamento Tocha 2019 0 40.32548,-8.81311

K
Table II (cont.). Number of galls of Trichilogaster acaciaelongifoliae detected during all post-release monitoring campaigns
CODE SITE
MONITORING
YEAR
Nº of DETECTED
GALLS (max
$
)
COORDINATES
COV Covões 2019 3 40.19463,-8.46630
FIG2 Figueira da Foz (McDonald´s) 2019 31 40.16622,-8.85358
FIG1 Figueira da Foz (Rotunda LIDL)
###
2019 81 40.15750,-8.84958
FIG Figueira da Foz 2019 5 40.14868,-8.83696
SOU Soure 2019 35 40.09979,-8.61969
PAT Paredes da Vitoria (Pataias) 2019 67 39.70796,-9.04883
STC Praia do navio, Santa Cruz 2019 5 39.14436,-9.37192
CAR Carapinheira, Mafra 2019 25 38.93549,-9.27610
CAP Praias da Costa da Caparica 2019 77 38.59994,-9.20743
ARR Estrada de Setúbal para praias 2019 0 38.49659,-8.93081
SAD Estrada Tróia - Comporta (RN Estuário Sado) 2019 23 38.42657,-8.82484
BRJ Brejinhos 2019 321 38.03127,-8.80838
PQS Pesqueiro Sancha 2019 11 38.02586,-8.82275
SIN2 Monte Feio - Sines 2 2019 2 37.99626,-8.84297
LSA Lagoas de Santo André 2019 818 37.99166,-8.85493
SIN1 Monte Feio - Sines 1 2019 6 37.98162,-8.84549
FAR Faro
##
2019 1117 37.02839,-8.00564
TOTAL 2016 66
TOTAL 2017 1124
TOTAL 2018 24793
(Sub)TOTAL 2019***** 6410
$
For some areas, more than one monitoring/year was performed; when this happened, the maximum number of galls
recorded was used for this table;
#
The area in Tocha burned in October 2017;
##
Although no galls were detected in
2018, some dry / old galls were observed in 2019, suggesting that they could be from the previous year and the
establishment could have occurred in 2018;
###
many trees were cut after releases; *In the end of 2017, 16 dried galls
were detected that had not previously been detected; **413 corresponds to the maximum number of galls detected in
3 monitoring during 2017 (1st 413; 2nd 304; 3rd 363); ***These 21 galls were detected only after conclusion of the
second report; **** 5690 galls were counted and the rest were estimated from observations. *****In 2019 it was no
longer possible to count the number of galls in sites with establishment in the previous years and as such (sub)Total
corresponds to the sum of galls counted only in sites with new establishment (2019), from wasps released in 2018,
from Portuguese populations.
From 2016 to 2018, in all sites where establishment occurred since 2016 (except for Perímetro Florestal das
Dunas de Cantanhede (Tocha) that burned) it was clear an exponential growth on the number of the galls
each year. This was expected since each female wasp can oviposit in average around 300 eggs, and therefore
when populations are established and (more) synchronized with the seasons and phenology of A. longifolia
in the Northern Hemisphere population growth can increase quite fast.
In addition to the increase in number of sites, the size of the areas/ range of dispersal in sites with
establishment since 2016 has also increased, with T. acaciaelongifoliae spreading into new areas, sometimes
several hundred meters from the focal trees where wasps were initially released (Figure 3). The furthest galls
detected were in Mata Nacional de Leiria (São Pedro de Moel) where some hundred galls were observed ca.
7km from the establishment site, close to Marinha Grande, and it is also in this site that the agent seems to
be spreading further, with the maximum distance between nearest plants with galls being around 800m in
2018. It was however not possible to know if this dispersal was achieve by natural means (i.e., the female

L
wasps flying) or aided by any human activity, e.g., wood logs transport or even intentional transport of some
galls.
Figure 3. Expansion of Trichilogaster acaciaelongifoliae gall population from 2016 to 2018 in the four sites (monitored in detail)
where establishment occurred in 2016: SJD- Reserva Natural das Dunas de São Jacinto, QUD- Mata Nacional das Dunas de Quiaios
(Dunas Quiaios), COI- Coimbra (Pólo II from University of Coimbra) and SPM- Mata Nacional de Leiria (São Pedro de Moel). a) Map
with recorded galls; b) Maximum distance between nearest plants with galls. Source: (López-Núñez et al., n.d.)
In three of the sites selected for “Monitoring” where galls were detected since 2016 and hence the number
of galls is in the order of thousands (not possible to count anymore; Mata Nacional das Dunas de Quiaios,
Reserva Natural das Dunas de São Jacinto and Mata Nacional de Leiria), the effects of T. acaciaelongifoliae
on A. longifolia were evaluated, analysing different parameters at vegetative (branch length, number of
secondary branches and number of phyllodes) and reproductive (number of pods) level (Figure 4). In each
site, 12 plants with galls were selected and similar number with no galls (the latter ones working as
Controls); in plants with galls, 5 branches with galls and 5 without galls were selected; in plants without galls,
5 branches were selected, totalizing 60 branches for each type of evaluation.
Although it is important to perform the evaluation of the effects of T. acaciaelongifoliae from the beginning
of the establishment, the number of plants with galls and the quantity of galls in each plant is still very small

Q
and sparse compared to the numbers of available floral and vegetative buds of A. longifolia, and
consequently the effects detected are expected to be indicative at this stage. Nevertheless, this monitoring
suggests that T. acaciaelongifoliae is promoting already some detectable effects (Figure 4). Comparing data
from 2018 and 2019, it was possible to observe that the number of secondary branches, the number of
phyllodes and the number of pods were all smaller in the branches of trees with galls, independently of
having or not galls in the measured branch (Figure 4), suggesting that the effects of T. acaciaelongifoliae are
not exclusively on the branches with galls, but at tree level, which could be expected since the formation of
galls represent a waste of energy for the all plant and not only for the branches where galls are developed.
As for the total branch length this increased in plants with galls (Figure 4), which may be the result of
secondary branches being prevented by gall formed at this level and as such apical growth occurs instead of
secondary branches.
Figure 4. Effects of Trichilogaster acaciaelongifoliae on Acacia longifolia at level of number of pods, number of secondary
branches, number of phyllodes and total branch length. Plants monitored included plants with galls developing (both branches
with T. acaciaelongifoliae galls and branches without galls) and plants with no galls (working as Controls). Bars represent the
percentage difference from 2018 to 2019 and are average numbers of the three of the sites with establishment since 2016: Mata
Nacional das Dunas de Quiaios, Reserva Natural das Dunas de São Jacinto and Mata Nacional de Leiria. Letters above bars are the
result of a Gaussian GLMM statistical test comparing the three types of branches; different letters above bars means that the
parameter measured is statistically different between branches. Source: (López-Núñez et al., n.d.)

As in previous years, amongst all the galls observed, some exhibited parts of A. longifolia phyllodes
(revealing that they were probably originated from oviposition in vegetative buds), but in 2019 most galls
were more probably originated from flowers buds (Figure 5), confirming the better synchrony between the
early stages of flower bud development and emergency of T. acaciaelongifoliae wasps. Although some galls
were observed with emergency holes along the year, only by May – July most galls were fully developed and
most wasps emerged. As in previous years, a few wasps were observed flying and ovipositing in the field,
with most galls revealing one or more emergency holes (depending on the number of cameras) indicating
that insects of T. acaciaelongifoliae must have emerged. Considerable variation was still observed in the
phenology of galls and sites suggesting that wasps did not emerge all at the same time and probably are not
taking the same time to develop the life cycle, raising some doubt about the number of generations per
year. As in the southern hemisphere, where T. acaciaelongifoliae takes around one year to complete the life
cycle, in Portugal the cycle seems to be approaching this length not only in the sites where establishment
occurred in 2016, but also in the ones where releases where done in 2018, i.e., with wasp more
synchronized with conditions (climate and phenology of A. longifolia) of the northern hemisphere.
Figure 5. Galls of Trichilogaster acaciaelongifoliae detected during post-release monitoring campaigns in 2019. Plant with
widespread galls (a), branch with many gall in more detail (b) and galls originating from flower buds (c).
Observations to detect galls of T. acaciaelongifoliae were also done in many non-target plants (please check
list of species in Annex 3), located in the areas surrounding A. longifolia trees where there is establishment
(a)
(b) (c)

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of the agent. Despite intensive search no (zero) galls of T. acaciaelongifoliae were observed in any non-
target species. These observations included Cytisus striatus (giesta-das-serras) and Acacia retinodes, the first
because in quarantine tests eggs were detected in the buds of this species, and the second because it has
commercial value in Italy and was later tested. Nevertheless, T. acaciaelongifoliae was not able to complete
the cycle in any of them, with no galls observed in quarantine (Marchante et al. 2011b, 2017).
3. Conclusions
Overall, there is establishment of T. acaciaelongifoliae along most of the Portuguese Coast and some more
inland sites, from Riba de Âncora, in the north, to Faro, in the south of the country, in a total of 32 sites; in
most of them, 2019 was the first year of establishment of T. acaciaelongifoliae (27 sites). During post-release
monitoring in Spring-Summer 2019, establishment was observed in 27 new sites, with almost 6500 first
generation galls being observed; counting of galls at sites with establishment from previous years was no
longer possible due to the high numbers. Most galls in sites with establishment since 2015 correspond
probably to fourth generation galls, although synchronization of T. acaciaelongifoliae with Northern
hemisphere conditions may be not complete, and as such we cannot be certain about the number of
generations. Nevertheless, independently of the number of generations, T. acaciaelongifoliae is establishing
and starting to spread by its own means in Portugal in at least 32 sites, despite several of this year new
populations still being very small. At one of the sites with establishment since 2015 (Mata Nacional de Leiria,
São Pedro de Moel), galls were observed ca. 7km from the establishment site, although it was not possible
to know if dispersion was done by natural means or aided by any human activity. Since the success of 2018
release was good, in 2019 releases were done in only one site.
It is noteworthy that while in the previous years the number of first generation galls/site was always low, at
most in the order of magnitude in the tens, in 2019 in several sites this number was much higher, sometimes
several hundred. This difference is most probably because the wasps released in 2015 (establishment in
2016) were of South African origin (life cycle was not synchronized with the season and phenology of
A. longifolia in Portugal), while in 2018 the releases were done for the first time with wasps originating from
Portuguese populations, and hence more synchronized not only with the phenology of the host-plant
(A. longifolia), but also with more adequate meteorological conditions.
Although the number of plants with galls and the quantity of galls in each plant is still very small and sparse
compared to the numbers of available floral and vegetative buds of A. longifolia in each site, the monitoring
of effects suggests that T. acaciaelongifoliae is reducing the number of secondary branches, phyllodes and
pods, not only on the branches with galls, but at tree level, reducing both reproductive and vegetative
growth of A. longifolia and consequently its invasive potential.
Depending on the monitoring and confirmation of establishment in the new sites, releases may continue or
stop in 2020. Monitoring of establishment of T. acaciaelongifoliae and its effects on A. longifolia will
continue.
While the team from the Centre for Functional Ecology (both from ESAC and UC) is committed to apply for
new funding, it is somewhat worrying that both projects under which release and monitoring campaigns are
being implemented (INVADER-IV and GANHA) will end by 2020 and funding to continue these campaigns is
not yet secured.

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4. References
Dennill, G.B., 1985. The effect of the gall wasp Trichilogaster acaciaelongifoliae (Hymenoptera: Pteromalidae) on
reproductive potential and vegetativa growth of the Acacia longifolia. Agric. Ecosyst. Environ. 14, 53–61.
Dennill, G.B., Donnelly, D., 1991. Biological control of Acacia longifolia and related weed species (Fabaceae) in South
Africa. Agric. Ecosyst. Environ. 37, 115–135.
Le Maitre, D.C., Gaertner, M., Marchante, E., Ens, E.J., Holmes, P.M., Pauchard, A., O’Farrell, P.J., Rogers, A.M.,
Blanchard, R., Blignaut, J., Richardson, D.M., 2011. Impacts of invasive Australian acacias: Implications for
management and restoration. Divers. Distrib. 17, 1015–1029. https://doi.org/10.1111/j.1472-4642.2011.00816.x
López-Núñez, F.A., Heleno, R.H., Ribeiro, S., Marchante, H., Marchante, E., 2017. Four-trophic level food webs reveal
the cascading impacts of an invasive plant targeted for biocontrol. Ecology 98, 782–793.
https://doi.org/10.1002/ecy.1701
López-Núñez, F.A., Marchante, E., Heleno, R.H., Duarte, L., Palhas, J., Marchante, H., n.d. Establishment, dispersal and
effects of the biocontrol agent Trichilogaster acaciaelongifoliae in Portugal. in prep.
Marchante, E., Kjøller, A., Struwe, S., Freitas, H., 2009. Soil recovery after removal of the N2-fixing invasive Acacia
longifolia: Consequences for ecosystem restoration. Biol. Invasions 11, 813–823. https://doi.org/10.1007/s10530-
008-9295-1
Marchante, E., Kjøller, A., Struwe, S., Freitas, H., 2008. Short and long-term impacts of Acacia longifolia invasion on the
belowground processes of a Mediterranean coastal dune ecosystem. Appl. Soil Ecol. 40, 210–217.
https://doi.org/10.1016/j.apsoil.2008.04.004
Marchante, H., Freitas, H., Hoffmann, J.H., 2011a. Post-clearing recovery of coastal dunes invaded by Acacia longifolia:
Is duration of invasion relevant for management success? J. Appl. Ecol. 48, 1295–1304.
https://doi.org/10.1111/j.1365-2664.2011.02020.x
Marchante, H., Freitas, H., Hoffmann, J.H., 2011b. Assessing the suitability and safety of a well-known bud-galling wasp,
Trichilogaster acaciaelongifoliae, for biological control of Acacia longifolia in Portugal. Biol. Control 56, 193–201.
https://doi.org/10.1016/j.biocontrol.2010.11.001
Marchante, H., Freitas, H., Hoffmann, J.H., 2010. Seed ecology of an invasive alien species, Acacia longifolia (Fabaceae),
in Portuguese dune ecosystems. Am. J. Bot. 97, 1780–1790. https://doi.org/10.3732/ajb.1000091
Marchante, H., López-núñez, F.A., Freitas, H., Hoffmann, J.H., Impson, F., Marchante, E., 2017. First report of the
establishment of the biocontrol agent Trichilogaster acaciaelongifoliae for control of invasive Acacia longifolia in
Portugal. EPPO Bull. 47, 274–278. https://doi.org/10.1111/epp.12373
Marchante, H., Marchante, E., Freitas, H., Hoffmann, J.H., 2015. Temporal changes in the impacts on plant communities
of an invasive alien tree, Acacia longifolia. Plant Ecol. 216, 1481–1498. https://doi.org/10.1007/s11258-015-0530-
4
Shaw, R., Schaffner, U., Marchante, E., 2016. The regulation of biological control of weeds in Europe – an evolving
landscape. EPPO Bull. 46, 254–258. https://doi.org/10.1111/epp.12308
Wilson, J.R.U., Gairifo, C., Gibson, M.R., Arianoutsou, M., Bakar, B.B., Baret, S., Celesti-Grapow, L., Ditomaso, J.M.,
Dufour-Dror, J.M., Kueffer, C., Kull, C.A., Hoffmann, J.H., Impson, F.A.C., Loope, L.L., Marchante, E., Marchante, H.,
Moore, J.L., Murphy, D.J., Tassin, J., Witt, A., Zenni, R.D., Richardson, D.M., 2011. Risk assessment, eradication,
and biological control: Global efforts to limit Australian acacia invasions. Divers. Distrib. 17, 1030–1046.
https://doi.org/10.1111/j.1472-4642.2011.00815.x

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Annex 1. FIRST REPORT – 2016

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Annex 2. SECOND REPORT – 2017

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Annex 3. THIRD REPORT – 2018

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Annex 4. FULL NAMES OF THE SITES REFERRED IN THE TEXT, FIGURES AND TABLES (2pages)
CODE SITE SHORT NAME SITE FULL NAME COORDINATES
ALH Alhadas (Cabecinho) Alhadas (Cabecinho), Figueira da Foz 40.174834,-8.787518
ANH Anha Anha, Viana do Castelo 41.674618,-8.801127
ANO Anobra Anobra, Condeixa-a-Nova 40.161626,-8.510096
ANT Antas Parque Natural do Litoral Norte (Antas) 41.61092,-8.80828
ARR Estrada Setúbal - praias Parque Natural da Arrábida 38.496597,-8.930805
BEZ Barrinha de Esmoriz Barrinha de Esmoriz, Esmoriz 40.963981,-8.647378
BRJ Brejinhos Brejinhos, Ermidas do Sado 38.01863,-8.4581
BZM Belazaima do Chão Belazaima do Chão, Águeda 40.527697,-8.330153
CAP Praias da Costa da Caparica
Paisagem Protegida da Arriba Fóssil da Costa da
Caparica (margem)
38.599935,-9.20743
CAR Carapinheira, Mafra Carapinheira, Mafra 38.935492,-9.276104
COI Coimbra (Pólo II) Coimbra (Pólo II from University of Coimbra) 40.18588,-8.41358
COI1 Coimbra (Patos) Coimbra (Patos) 40.209237,-8.401237
COV Covões Covões, Coimbra 40.194632,-8.466302
EIX Eixo Eixo, Aveiro 40.620281,-8.568342
EIX2 Eixo (Quinta de S. Francisco) Eixo (Quinta de S. Francisco), Aveiro 40.616127,-8.567075
ESP Esposende Parque Natural do Litoral Norte (Esposende) 41.508999,-8.784351
EST Coimbra (ESAC) Coimbra (ESAC, greenhouse) 40.216722,-8.450210
FAR Faro Parque Natural da Ria Formosa (Ludo, Faro) 37.028391,-8.005642
FIG Figueira da Foz Figueira da Foz 40.148682,-8.836959
FIG1
Figueira da Foz (Rotunda
LIDL)
Figueira da Foz (Rotunda LIDL) 40.157502,-8.849582
FIG2 Figueira da Foz (McDonald´s) Figueira da Foz (McDonald´s) 40.166223,-8.853586
GAL Gala, Parque de Merendas Gala, Parque de Merendas, Figueira da Foz 40.123295,-8.859909
HEL Condeixa Condeixa-a-Nova 40.113693,-8.513775
IP3 Acesso IP3 Acesso IP3, S. Miguel do Outeiro, Viseu 40.586589,-8.015036
LAG Lagoa da Vela
Mata Nacional das Dunas de Quiaios (Lagoa da
Vela)
40.269481,-8.799287
LAV Lavos
Mata Nacional das Dunas da Costa de Lavos
(Lavos)
40.097376,-8.856496
LEI Leirosa Mata Nacional das Dunas da Leirosa (Leirosa) 40.076190,-8.865008
LSA Lagoas de Santo André
Reserva Natural das Lagoas de Santo André e da
Sancha
37.991658,-8.854934
MIR Mira
Perímetro Florestal Dunas e Pinhais de Mira
(Mira)
40.394569,-8.789050
MOR Morraceira Morraceira, Figueira da Foz 40.139481,-8.844876
MUR Mata do Urso Mata Nacional do Urso 39.983285,-8.914007
PAT Paredes da Vitoria (Pataias) Paredes da Vitoria (Pataias) 39.707963,-9.048827
PED Pedrogão Mata Nacional do Pedrogão 39.937940,-8.927712
PEN Quinta Pentieiros Quinta Pentieiros, Viana do Castelo 41.775557,-8.657753
POC1 Pocariça (Cantanhede1) Pocariça (Cantanhede1) 40.382712,-8.574368
POC2 Pocariça (Cantanhede2) Pocariça (Cantanhede2) 40.37305,-8.579074
PQS Pesqueiro Sancha
Reserva Natural das Lagoas de Santo André e da
Sancha (Pesqueiro Sancha)
38.02586,-8.82275
QUD Dunas de Quiaios
Mata Nacional das Dunas de Quiaios (Dunas
Quiaios)
40.22476,-8.88622
QUP Quiaios N
Mata Nacional das Dunas de Quiaios (Pinhal
Quiaios)
40.241645,-8.854573
(continues next page)
FULL NAMES OF THE SITES REFERRED IN THE TEXT, FIGURES AND TABLES (cont.)

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CODE SITE SHORT NAME SITE FULL NAME COORDINATES
RBA Riba de Âncora
Perímetro Florestal de Serras de Vieira e Monte
Crasto (por confirmar) - Riba de Âncora (Baldios
de Riba de Âncora)
41.808889,-8.796121
SAD Estrada Tróia - Comporta
Reserva Natural do Estuário do Sado (Estrada
Tróia - Comporta)
38.426573,-8.824844
SBV Serra da Boa Viagem
Mata Nacional do Prazo de Santa Marinha (Serra
Boa Viagem)
40.20037,-8.88969
SEI Seixo
Perímetro Florestal Dunas e Pinhais de Mira (por
confirmar; Seixo, Vagos)
40.497200, -8.754436
SIN1 Monte Feio - Sines 1 Monte Feio - Sines 1 37,981898,-8,845609
SJD Dunas de São Jacinto Reserva Natural das Dunas de São Jacinto 40.672717,-8.740729
SOU Soure Soure (RAIZ - Pai Daniel) 40.099786,-8.619687
SPI Espinho
Perímetro Florestal Dunas de Ovar (por confirmar,
Maceda, Espinho)
40.923320,-8.658262
SPM São Pedro de Moel Mata Nacional de Leiria (São Pedro de Moel) 39.75711,-9.02338
STC Praia do navio, Santa Cruz Praia do navio, Santa Cruz 39.144362,-9.371916
TOC Tocha
Perímetro Florestal das Dunas de Cantanhede
(Tocha)
40.34837,-8.81704
TOC1 Tocha N
(Tocha Norte)
40.320083,-8.845127
TOC2 Tocha (Caniceira)
(por confirmar, Caniceira, Tocha)
40.3476,-8.77368
TOC3 Cruzamento Tocha
(cruzamento Tocha)
40.3254768,-8.8131098
TOC4 Charco Berlengas
(por confirmar, Charco Berlengas)
40.32656634,-8.7821874
VAG Dunas Vagos (GANHA)
Perímetro Florestal das Dunas de Vagos (por
confirmar, Dunas Vagos)
40.535932,-8.74104
VIV Vila Verde Vila Verde, Figueira da Foz 40.154741,-8.795803
VLE Vieira de Leiria
Mata Nacional de Leiria (por confirmar, Vieira de
Leiria)
39.865863,-8.971235
VNR Vila Nova da Rainha Vila Nova da Rainha, Tondela 40.47055,-8.09458
VNMF Vila Nova de Mil Fontes
Vila Nova de Mil Fontes (Praia das Furnas,
included in Parque Natural Sudoeste Alentejano e
Costa Vicentina)
37.714934, -8.7845
NOTE: for some sites it was not possible to verify if their limits were inside the Matas Nacionais or Perímetros Florestais
referred.

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