Another Chapter in the History of the European Invasion by the Western Conifer Seed Bug, Leptoglossus occidentalis: The Iberian Peninsula

Round 1

Reviewer 1 Report

The overall aims of the authors were to describe the genetic diversity and invasion pathways of N. American Western conifer seed bugs in the Iberian Peninsula. The authors collected bugs from 14 sites, and used a number of different markers to look at genetic diversity. The data suggest at least 3 introduction sites. The methods were based on established methods from a 2019 study and expanded upon that dataset to focus on the Iberian Penninsula.

The paper addresses an important question, and the findings of multiple invasion pathways are disturbing and suggest more needs to be done to curb the spread of this insect. The paper is well written and well laid out and the methods seem sound except for the inclusion of very low numbers of specimens at several sites – the authors should justify the inclusion of these.

The paper could be strengthened and its appeal broadened by a brief discussion of the implications of the findings for international trade policy – e.g. description of the legislation in place to prevent beetle spread and how these policies may be failing, implications for other geographical areas in which this bug is not present, and the possibilities of future pests travelling along the same pathway.

L63 genus should be genera

L76-78 rephrase - unclear

L91-92 rephrase – unclear

L98-112 cut down if possible – long summary of another paper

L119 could expand here about how better understanding invasion pathways would help stop establishments of other non-native species

L122 beetle samples?

Table 1 Lesieur et al 2019 only used sites for which at least 3 specimens were collected – I cannot comment on whether it is appropriate to use < 3 as in 3 of the sample sites

L320 Eastern

L485-487 rephrase

L489 hitchhike?

Author Response

Reviewer 1

The overall aims of the authors were to describe the genetic diversity and invasion pathways of N. American Western conifer seed bugs in the Iberian Peninsula. The authors collected bugs from 14 sites, and used a number of different markers to look at genetic diversity. The data suggest at least 3 introduction sites. The methods were based on established methods from a 2019 study and expanded upon that dataset to focus on the Iberian Penninsula.

The paper addresses an important question, and the findings of multiple invasion pathways are disturbing and suggest more needs to be done to curb the spread of this insect. The paper is well written and well laid out and the methods seem sound except for the inclusion of very low numbers of specimens at several sites – the authors should justify the inclusion of these.

The paper could be strengthened and its appeal broadened by a brief discussion of the implications of the findings for international trade policy – e.g. description of the legislation in place to prevent beetle spread and how these policies may be failing, implications for other geographical areas in which this bug is not present, and the possibilities of future pests travelling along the same pathway.

R: Thank you for your constructive comments on our manuscript. We have taken them into account and modified our manuscript accordingly.  Please see below our answers to your comments.

L63 genus should be genera

R: Done

L76-78 rephrase – unclear

R: We rephrased as follow “The vast majority of the world producing countries are located in the Mediterranean Basin (e.g. Italy, Turkey, Lebanon and Tunisia; Carrasquinho et al. 2017), with almost 70% of the plantations of P. pinea in the world located in the Iberian Peninsula (Portugal, and Spain), making the western Mediterranean region the major producer of this valuable seed product (EUFORGEN 2009). Therefore, it is not surprising that most of the studies targeting this pest in the Mediterranean Basin are focused on P. pinea highlighting its economic damage (Bracalini et al. 2015; Mutke and Calama, 2016; Calama et al. 2017; Farinha et al. 2018a, 2018b, 2021).” Lines 72-82

L91-92 rephrase – unclear           

R: We rephrased as follow “Furthermore, the complexity of tracing its invasion routes increases as adults aggregate during the autumn in human-made structures or in forest refuges, such as under the bark of coniferous trees (Lesieur 2014). This behaviour can favour the mixing of individuals from different populations which may lead to a genetic homogenisation of the populations making it difficult to reconstruct the history of the invasion (Boubou et al. 2012).” Lines 95-101

L98-112 cut down if possible – long summary of another paper

R: This paper was the first one assessing the invasion pathways of Leptoglossus occidentalis in Europe. In our manuscript, we are using part of their dataset but also basing our analyses and interpretations from their findings. We believe it is of importance to summarise them in the introduction to the readers for a better understanding of our study and what it is adding. We have kept our summary but tried to shorten it a little as follow: “Populations from the native western North America were introduced to the eastern part of North America from where multiple, independent, introductions to Europe occurred, acting as a bridgehead (i.e. an invasive population serving as a source for others; Lombaert et al. 2010). These introductions, combined with translocation of individuals within the European continent through human-mediated transportations as hitchhikers were also suggested to have favour the insect’s rapid colonization (Lesieur et al. 2019).” Lines 103-110

L119 could expand here about how better understanding invasion pathways would help stop establishments of other non-native species

R: We added the following sentence: “Assessing invasion pathways of such species will help identify factors driving invasions in order to develop appropriate management strategies to avoid further introductions and spread of non-native species (Allendorf and Lundquist 2003; Bock et al. 2015; Bras et al. 2022).” Lines 124-126

L122 beetle samples?

R: We added “we analysed samples of the Western conifer seed bug from the Iberian Peninsula” line 131

Table 1 Lesieur et al 2019 only used sites for which at least 3 specimens were collected – I cannot comment on whether it is appropriate to use < 3 as in 3 of the sample sites

R: It is common for studies using mitochondrial DNA to include sites where less than 3 samples were collected (eg. Bras et al 2019, Javal et al 2019, Dzurenko et al 2021, Valentin et al 2017). It provides information regarding the geographic distribution of haplotypes and permits to describe the genetic diversity of a given species. Most of the analyses of COI requires at least more than 2 individuals. However, the Analysis of molecular variation (AMOVA) for instance, doesn’t require a specific number of individuals per site as we are grouping sites together based on chosen criteria.

L320 Eastern

R: Done

L485-487 rephrase

R: We rephrased it as follow: “Our study provides evidence of multiple introduction events of the Western conifer seed bug in the Iberian Peninsula which were likely favoured by human activities. This genetic evidence combined with first record dates suggest that stratified dispersal mechanism, characterised by the insect’s and human-mediated dispersal, better explain the history of L. occidentalis invasion in this region. Indeed, the intensive trade of cones of P. pinea in Iberia and the high capacity of this insect to hitchhike is probably leading to human-mediated dispersal.” Lines 504-511

L489 hitchhike?

R: Corrected

Reviewer 2 Report

Scenario development of a scenario for the dynamics of invasion of L. occidentalis in the Iberian Peninsula and identification of ways for the pest to invade the Iberian Peninsula, based on the original features of mtDNA and microsatellites identified by the authors, are of significant scientific interest. The conclusion that there were at least three invasions of L. occidentalis into Iberia and the absence of a new introduction directly from western North America is quite reasonable. Identification of clusters in the genetic diversity of the secondary range of the invader on the Iberian Peninsula provides an understanding of the history of invasion and the mechanism of the stratified distribution of the species in this territory.

I recommend the manuscript for publication.

Comments for author File: Comments.docx

Author Response

Thank you for your very positive comment on our manuscript.

Reviewer 3 Report

The article deals with the spread of the invasive pest Leptoglossus occidentalis in the Iberian Peninsula. It is a serious pest of conifer seeds and one of the fastest spreading invasive pest in Europe.

The article is suitable for publication after minor revision.

In particular, authors should use the citation template provided in the author guidelines in the MDPI. Then they would avoid errors in both in-text citations and references.

For example:

L51 is a citation of Clavero & García-Berthou, 2005 but in the references is 2006

L52 is a citation Che et al. 2006 but this citation isn´t in the references

L68 is Roversi et al. 2009 but this isn´t in the references, only Roversi 2009

L420 is Handley et al. 2011 probably to be Lawson Handley et al. 2011 as in L81

L434 is Koeber 1963 but this citation isn´t in the references

Then there are inconsistencies in references such as the use "and" and "," between names, see L513, L515 and perhaps L517

In addition, the word "Iberian" should be added before the word "Peninsula" in L343, 409, 441 and 491

the word "Peninsula" should be added after the word "Iberian" in L445

In L410 it says that, multiple introductions is a common phenomenon, but there are known cases in Europe where there has only been a single introduction. The authors should mention this option in the discussion. The relevant literature is:

Faccoli M, Simonato M, Rassati D 2016: Life history and geographical distribution of the walnut twig beetle, Pityophthorus juglandis (Coleoptera: Scolytinae), in southern Europe. Journal of Applied Entomology 140: 697-705.

Dzurenko M, Ranger CM, Hulcr J, Galko J, Kaňuch P 2021: Origin of non-native Xylosandrus germanus, an invasive pest ambrosia beetle in Europe and North America. Journal of Pest Science 94: 55-562.

Author Response

The article deals with the spread of the invasive pest Leptoglossus occidentalis in the Iberian Peninsula. It is a serious pest of conifer seeds and one of the fastest spreading invasive pest in Europe.

The article is suitable for publication after minor revision.

R: Thank you for your positive feedback on our manuscript and your constructive comments. We have modified our manuscript accordingly.

In particular, authors should use the citation template provided in the author guidelines in the MDPI. Then they would avoid errors in both in-text citations and references.

For example:

L51 is a citation of Clavero & García-Berthou, 2005 but in the references is 2006

R: Corrected

L52 is a citation Che et al. 2006 but this citation isn´t in the references

R: Corrected. The text reference was missing an “n”. The correct citation is “Chen et al. 2006”.

L68 is Roversi et al. 2009 but this isn´t in the references, only Roversi 2009

R: This citation was removed from the text

L420 is Handley et al. 2011 probably to be Lawson Handley et al. 2011 as in L81

R: Citation on L420 was corrected

L434 is Koeber 1963 but this citation isn´t in the references

R: Corrected. The citation was added to the bibliography list (citation #84).

Then there are inconsistencies in references such as the use "and" and "," between names, see L513, L515 and perhaps L517

R: We also uniformed the style in all the manuscript and followed Diversity journal’s guideline regarding references.

In addition, the word "Iberian" should be added before the word "Peninsula" in L343, 409, 441 and 491

the word "Peninsula" should be added after the word "Iberian" in L445

R: Done

In L410 it says that, multiple introductions is a common phenomenon, but there are known cases in Europe where there has only been a single introduction. The authors should mention this option in the discussion. The relevant literature is:

Faccoli M, Simonato M, Rassati D 2016: Life history and geographical distribution of the walnut twig beetle, Pityophthorus juglandis (Coleoptera: Scolytinae), in southern Europe. Journal of Applied Entomology 140: 697-705.

Dzurenko M, Ranger CM, Hulcr J, Galko J, Kaňuch P 2021: Origin of non-native Xylosandrus germanus, an invasive pest ambrosia beetle in Europe and North America. Journal of Pest Science 94: 55-562.

R: We mentioned this phenomenon earlier in our discussion. We modified our sentence to clarify this point and added Dzurenko references. The other reference was not ruling out the possibility of multiple introductions so we didn’t include it. “However, more and more studies using neutral molecular markers are showing that such species are not necessary presenting significant loss of genetic diversity, often resulting from multiple introductions in the novel range, or thea single introduction of a large number of individuals (Facon et al. 2008; Ciosi et al. 2008; Kolbe et al. 2013; Javal et al. 2019; Dzurenko et al, 2021; Bras et al. 2022). “ Lines 373-376