Mangrove forests possess multiple functions for the environment and society through their valuable ecosystem services. Along with this, the mangrove forests have large and diverse social values, in combination contributing to the health and wellbeing of the surrounding communities. This study aims (i) to assess the benefits of mangrove forests and their impact on subjective and psychological wellbeing of coastal communities and (ii) to understand the challenges coastal communities face that limit sustainable wellbeing. We have used a mixed methodological approach, combining workshop, interview, and survey, to obtain qualitative and quantitative information from two coastal communities in Malaysia and Indonesia. For quantitative data, 67 participants from both coastal communities participated using a pre-tested structured questionnaire. To obtain opinions from key informants in Malaysia and Indonesia, we organized two stakeholders' workshops and community interviews. When merging these interviews and workshops, we identified the following three themes related to the perception of mangrove forest benefits: (1) the advantage of living in a natural countryside; (2) the natural resources supporting employment, income, and family security; and (3) the increase in subjective and psychological wellbeing. The mean score of wellbeing for Indonesian participants (28.6) was slightly higher than that for Malaysian participants (26.2) and was significant. Overall, the respondents felt happy because the combination of job security and leisure activities supports feeling content and satisfied. The analyses also suggest that the combination of exposure to coastal environments and stress reduction promotes good mental health; however, diagnostic health data are lacking. The lower score of mental wellbeing in Malaysia is attributed to respondents involved in risky fishing activities and local regions with excessive tourism. The findings from this study imply that coastal mangrove forest management plays an important role in the living conditions of coastal communities and their subjective and psychological wellbeing. Hence, restoration and sustainability of mangrove ecosystem are important. Mangrove forests possess multiple functions for the environment and society through their valuable ecosystem services, including provisioning, regulating, habitat, and cultural services. These unique forests bordering tropical coastlines worldwide –3 have hSubjective wellbeing is a multidimensional construct capturing basic human psychological needs, such as security, materials supporting a satisfactory life, health, and successful social relationships . It is kThere has been substantial research on the linkages between subjective wellbeing and nature in many countries, particularly in western nations, including Australia, East Asia, European countries, and North America , 27. MorTo the best of our knowledge, no previous studies on subjective wellbeing effects of forests or nature specifically explored the impact of mangrove forests on psychological wellbeing. As mentioned above, having these research gaps, this study aimed (i) to assess benefits of mangrove forests and their impact on subjective and psychological wellbeing of coastal communities and (ii) to understand the challenges toward sustainable wellbeing of coastal communities. Data for this study were collected from two coastal communities in Malaysia and Indonesia. We approach subjective wellbeing as an assessment based on personal judgments of general happiness or satisfaction  and psycResearch to date has partly tested the benefits of mangrove ecosystem on subjective and psychological wellbeing of coastal communities. We assumed that mangroves through their direct  and indirect  benefits influence both subjective and psychological wellbeing of coastal communities . In thisTwo mangrove sites were selected based on the richness of the mangrove ecosystem in the two Southeast Asia countries of Malaysia and Indonesia.Matang Mangrove Forest Reserve (MMFR)is located in the north-west coast of the Peninsular of Malaysia (4°51′7.14″N−100°38′48.50″E). The Perak State Forestry Department manages MMFR covering about 40,466 ha and still considered as the best managed mangrove forest in the world is a protected mangrove area in Surabaya, Indonesia (7°18′76″S−112°48′ 922″ E to 7°18′328″ S−112°50′691″E). MWS covers ~700 ha and is managed by the Department of Food Security and Agriculture of the City of Surabaya Government and declared as a conservation area by the City Major of Surabaya under Surabaya City Regional Regulation No. 3, 2007  from 22 organizations joined in the workshops, including: the Department of Forestry, Department of Fisheries, Perak Fishermen's Association, Fisheries Research Institute Malaysia, Malaysian Nature Society of Perak Branch, Maritime Institute of Malaysia, Malaysian Wood Industries Association, Wetlands International Malaysia, Forest Research Institute Malaysia, Department of Survey and Mapping (Perak), Regional Planning and Development Agency (East Java and Surabaya), Fishery and Marine Department of East Java, communities leaders, NGOs, universities, local administrations, and others.Key informant interviews are qualitative in-depth interviews with a wide range of people who have first-hand knowledge about the coastal communities. We conducted 67 interviews with the coastal communities  at both sites.Quantitative data on socio-demographic characteristics and subjective and psychological wellbeing were collected from the respondents. We used a purposeful structured questionnaire for collecting quantitative data on subjective wellbeing of two core components to explore the relationship between experiences and reactions toward the mangrove ecosystem of coastal communities and their subjective wellbeing. For psychological wellbeing, we followed the Warwick–Edinburgh Mental Wellbeing Scale (SWEMWBS), which is being widely used to understand psychological wellbeing of community people –41, 57.Through the community leaders, we interviewed 67 respondents (40 in MMFR and 27 in MWS) who agreed to participate. All interviews were conducted through phone due to mobility restrictions during COVID-19 pandemic. As English is not the first language of the respondents, the questions were initially developed in English and then translated to Malay, the main language used by the local communities in Matang, Malaysia, and Surabaya, Indonesia.Ethical approval was obtained from the research ethics committee of the School of Social Sciences Ethics Committee, Heriot-Watt University. In addition, we secured respondents' verbal consent for participation and audio-recording for this study. All respondents remain anonymous in this study.All qualitative data from the interview transcripts were transcribed and translated into English for data analysis. We used the inductive approach of thematic analysis introduced by Braun and Clarke , which dFor quantitative data, SWEMWBS scores from respondents were calculated to give a mean score within and between the population sample from Malaysia and Indonesia, respectively. Mean scores for MMFR and MWS were compared according to the score categorization suggested by Warwick Medical School , 60. DesRespondents in both MMFR and MWS reported several direct and indirect benefits from mangrove forests . In MMFRThe thematic analysis of qualitative data revealed the following three themes: (1) the advantage of living in a natural countryside; (2) the natural resources supporting employment, income and family security; and (3) the increase in subjective and psychological wellbeing. Below, we outline some features of these three themes:Theme 1: The advantage of living in a natural countrysideI feel that it is more relaxing here because places like fishing village are always more relaxed.” Furthermore, respondent-015: “Since we are living in the countryside, the pace of living here is slower, not like in the city where the pace of living is faster.”Respondent-013:“I think you can see changes, changes from the economy in one family.”This may illustrate the more laid-back lifestyle in MMFR compared with urban areas that leads to carefree spirit among local communities. Meanwhile, respondent-006 attributed the relaxed and carefree spirit among local communities to the improving economic situation in MMFR: “Theme 2: The natural resources supporting employment, income, and family securityNatural resources such as wood for charcoal and fisheries have provided support for the livelihood of local communities. Charcoal production has created economic opportunities for small businesses and job opportunities in factory. Many respondents sell charcoal and other products made from charcoal, such as wood vinegar for a living. Mangrove forests in MMFR provide a breeding ground for fisheries which actively contribute to the abundant fisheries resources. For example, shrimp is one of the main fisheries catch in Kuala Sepetang.we supply it (shrimp) to the wet market” and “it is only here and the nearby areas.”Respondents commented that “Local communities can secure basic needs for their daily life due to the improving economic situation in Matang. Most of the respondents showed satisfaction of their current earnings that can be mainly attributed the satisfaction toward being able to support their livelihood.we are fine with current income sources, it's sufficient for villagers like us” and “It is enough for our spending.” With Kuala Sepetang being located in the countryside,For instances, respondents commented that “t is sufficient to support a living in countryside but not enough for a living at the city.”Respondent-015 mentioned that “iMangrove forests are the natural habitat for many species that are important to keep the food cycle in the ecosystem  and hencThemes 3: The increase in subjective and psychological wellbeingRespondents in MWS also mentioned that they felt happy because there are job security and leisure activities which provide an environment which supports a comfortable lifestyle.Tourism activities provide jobs for many of the local people in this area. We feel so happy because these jobs can make our economic status get better.”Respondent-026 commented that her husband enjoyed this place as her husband loved fishing. Another respondent-021 also revealed: “Mangroves contribute to the tourism industry with various activities to offer, including nature education center, place for bird or fireflies watching and river cruises . Eco-touWe investigated two main variables, namely, satisfaction with life and work and feeling happy, to assess the subjective wellbeing of respondents. We asked four questions related to satisfaction with life and work. The results show that more than 70% of the respondents were satisfied with their life and work in coastal areas . Iqbal  reportedU = 748.00, z = 2.67, p < 0.05).The results of psychological wellbeing of respondents due to the presence of mangrove forests showed that the mean scores of SWEMWBS for Indonesian respondents  are higher than those for Malaysian respondents . A non-parametric (Mann–Whitney) test was conducted to examine the differences between mean scores for Malaysian and Indonesian respondents to accommodate for the unequal number of participants in both study areas. The analysis shows that the differences between mean scores of SWEMWBS for Indonesia and Malaysia were significant , average mental wellbeing (20–27), and low mental wellbeing (7–19). This outcome of mental wellbeing is associated with the neighborhood mangrove environment in which coastal communities of Matang and Wonorejo live. White et al. , 65 suggThe lower score of mental wellbeing in MMFR is attributed to the involvement of respondents in risky fishing activities. Fishing is known to be dangerous, at times, and can be exposed to multiple risks, including weather and sea conditions , 68. In Representatives in the workshops held in both study sites identified several challenges that may jeopardize the benefits of mangrove forests. They also suggested various mitigation measures toward sustainable management of mangrove forests and enhancement of community development. Participants expressed that MMFR was well managed specifically in terms of timber production for charcoal. Despite having good management plans, one of the main concerns regarding the MMFR management plans shared by the participants was risk of infrastructure development and expansion of urbanization in forest areas along with incidences of illegal logging, forest degradation, and funding constraints for forest rehabilitation . RepreseParticipants in the Indonesian workshop reported that MWS is a newly declared mangrove forest reserve, and all resources were being managed by a single government agency, the “Food and Agriculture Agency.” Being a new forest reserve, local people were not very much aware about resource conservation, and they were not involved in the management. Participants identified limited funding opportunities for MWS management. They also reported river pollution and lack of educational resources for visitors and local guides as challenges. They suggest various measures for mitigating these issues. Mangrove forests have many benefits for coastal communities of the MMFR and MWS in terms of socio-economic opportunities which directly or indirectly impact the subjective and mental wellbeing of coastal communities. Respondents from both study areas identified the development of the tourism sector as one of the primary economic opportunities for local communities with the creation of diverse job opportunities in tourism, accommodation, and catering. Albeit the main income opportunity in MMFR is related to charcoal industries, other opportunities have been identified, including tourism and fisheries. In MWS, the opportunities stemmed from tourism industries where participants worked as staff and food sellers. However, the coastal communities in MMFR and MWS are also aware of challenges that could possess threats to their wellbeing and livelihoods, driven by declining yields for fisheries over the years, rubbish pollution, inconsistencies in the rehabilitation programme for the mangrove forest, and a general lack of mangrove management staff.Respondents from both regions have high to average mental wellbeing based on SWEMWBS scores. This outcome shows that the benefits provided by mangrove ecosystems lead to stress reduction when economy resources and job opportunity are secure, and a good mental health of local communities. The difference in the SWEMWBS mean scores between both study sites is rather small among the participants and likely attributed to differences in the nature of industry in the coastal communities . Thus, proper mangrove forest management plays an important role in safeguarding and developing subjective and psychological wellbeing of coastal communities through ensuring the availability of long-term benefits provided by mangrove forests and co-ownership/active engagement in future development plans and implementation.One of the main limitations of this study was a small number of interviews, which might affect the generalization of results. Future research may focus on having a larger number of interviews to provide a statistically reliable analysis. Our research only assessed the respondents' perception on benefits and did not quantify the benefits. Adding an economic valuation of ecosystem services would provide a more complete evaluation of mangrove ecosystem services, requiring further studies. In addition, the association between coastal communities' subjective and psychological wellbeing, and the mangrove ecosystem are under study. Such a relationship is pertinent for coastal communities to improve and sustain contentment and life satisfaction. Hence, this study warrants stakeholders and scholars to explore factors that associate for better wellbeing of coastal communities.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Heriot-Watt University, Social Sciences Ethics Committee. The patients/participants provided their written informed consent to participate in this study.G-NK, IU, TW, AS, AA, and TN conceived of the idea and apply for research funding. G-NK, IU, TW, AS, AA, TN, JN, and LM developed the workshop and interview materials. G-NK, IU, JN, and LM performed data collection and data analysis. All authors discussed the results and contributed to the final manuscript.This research was funded by Global Challenges Research Fund, The Scottish Funding Council, grant number SFC: P20GCRF7.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The data comprise three corpora consisting of three media programs from China, the US, and the UK. Results show that there is a statistically significant difference in the frequency of the DM so and the DM and, with each DM having the highest frequency in a specific corpus. Four co-occurring strings  are identified in the three corpora with the DM co-occurrence “and so” having the highest frequency in the American program, supporting the claim that this combination is a typical use in American English. The general positional distribution of the three DMs is similar with the highest tendency in the initial position, which can be attributed to the program’s interactivity. The findings will enhance our understanding of the three DMs used in media discourse and should be of practical significance to media hosts and guests in achieving better bilateral communication.This article, which is part of an on-going large-scale study, quantitatively explores and compares the frequency, patterns, and positions of the three most frequently used discourse markers (DMs):  Lexical corpora  such as Against this backdrop, the current study aims to explore media talk, specifically TV interviews. Media talk, as a particular genre, provides insights into the nature of mass communication and serves as a bridge between the media, public opinion, and public knowledge. Recently, media talk has begun to be studied as a phenomenon in its own right . StudiesThe characteristics of the program reveal the use of a set of pragmatic language realizations, such as the use of discourse markers (DMs) . DMs canIn the past, there has been a great deal of interest in the theoretical study of DMs, with much of that interest focusing on their definition, meaning, and functions. For instance, the majority of the studies tend to advocate for different interpretations of DMs, such as discourse connectives , discourhát in semi-guided informal conversations and job interview dialogues; I mean, of course, oh, well, I think, you know) in BBC and CNN political interviews; well in a spoken learner corpus between NSs and NNSs.Previous research on the three selected DMs has also examined their functions in structural relations, cohesive relations, and interactional relations, particularly their role in achieving discourse coherence. These studies use either natural data, such as sociolinguistic group interviews in which several people are invited to prompt one another to speak , a film so can be classified into two types. One is the investigation of the multifunctionality of the DM so in various contexts, and the other is primarily employing the comparative approach. The exploration of the function of the DM so has been conducted in learner corpora  and British National Corpus (BNC) and discovers that the function and frequency of so vary according to text genre. so in the LINDSEI-AR sub-corpus. Some comparative studies on the DM so in learner corpora are also reported in and tend to rely on loosely extracted examples from multiple sources, such as examples retrieved from the spoken learner corpora  of the three DMs across the three corpora in terms of the above-mentioned aspects?The Point with Liu Xin has been selected as the Chinese TV interview. The data of this interview consist of two episodes, which are together referred to as the Chinese Corpus. Amanpour and Company is a global-news interview program on public broadcasting service (PBS). Two episodes are selected as the sample data, termed the US Corpus. HARDtalk is a BBC television and radio program that airs on BBC News Channel. Likewise, the data from this interview also comprise two episodes and is coded as the UK Corpus. The data for this study are randomly extracted from an on-going large-scale project of 120 episodes . The composition of the three corpora is shown in The study uses three corpora of TV interviews from China, the US, and the UK. One representative program is chosen from each of the three countries. The three programs are highly representative with a combination of global vision and unique local characteristics, in which celebrities from various fields are interviewed. Each of the three programs begins with a concise introduction with some background material, and then they move on to the conversation with challenging questions and discussions; the total running time of each episode is no more than 30 mins. The corpus method complements quantitative and qualitative approaches and often works well and effectively in conjunction with them . The appso, and, but in the form of concordance lines in the three corpora can be drawn. The corresponding generated concordance lines are saved for subsequent analysis. The frequency of detected DMs is calculated through statistical procedures such as the calculation of the absolute frequency of the three DMs, which include both DMs and non-DM uses, and then the DM frequency is calculated in line with so is a pro-form (I guess so), degree adverb (so good), or in fixed patterns (so…that); if and connects elements below the clause level; if but in fixed expressions .The corpora were built for the examination of the frequency, patterns, and position of the three selected DMs, due to the importance of the spoken corpus in DM research . First, via the filter function. The co-occurrences can also be visualized using the GraphColl tool, through which collocations are identified and displayed in a collocation graph, which visualizes the collocates’ strength, frequency, and position. The combination of the three DMs can be generated by excluding the non-DM clusters. Using the filter and GraphColl function, the collocation frequency is identified, enabling the investigation of the co-occurrence of the three DMs.Second, all the concordance lines registering with the co-occurrence of the three DMs are extracted But, we arrived on time.), medial position , and final position . Using the KWIC function, the positional distribution of the three DMs in particular concordances in the three corpora was extracted, and their frequency was calculated.Third, the positional distribution of the three DMs can be observed and counted using the search results for DM frequency. The operational definition of DM position is in line with Finally, regarding the comparison of the frequency of the three DMs across the three corpora, normalization is used to allocate the frequency of the specific word to a common basis . Counts so, and, but across the three corpora. The log-likelihood statistic is preferred in frequency comparisons between corpora, as demonstrated in The frequency ranking can be calculated by comparing the normalized frequency. The log-likelihood test was used to determine whether there is a significant difference in the frequency of the DM so in the Chinese Corpus. Forty-five instances are used as DMs, while the other 15 instances are non-DM. The DM and occurs 162 times in the corpus, of which 82 instances are excluded; hence, and as DM occurs 80 times. There are 31 instances of but in total, of which four are excluded; thus, but as DM occurs 27 times. The frequency of the three DMs in the Chinese Corpus is shown in There are 60 instances of so occurs 55 times in total, of which 37 are used as a DM. The DM and occurs 253 times in total, with 155 of those instances being used as a DM. There are 29 instances of but in the corpus, except for six cases; thus, but as DM appears 23 times.In the US Corpus, the DM so in the UK Corpus, 22 are used as DMs when the remaining nine instances are excluded. There are 182 instances of and, of which 87 instances are used as a DM after excluding 95 cases. There are 54 cases of but, with 47 cases used as DM when the other seven non-DM uses are excluded.Altogether, “so but,” “and so”) that each occurs only once. In other words, the DM cluster “and so” is an example of EDM-IDM, while “so but” is a DM cluster of IDM-CDM. The visualization of the combination of “so but” is shown in The patterns discussed in the present study are the co-occurrence/combination of DMs or DM clusters. The frequency of the combination of the three selected DMs in the Chinese Corpus can be seen in “and so”), one example of CDM-IDM co-occurrence of (“but so”), and one instance of the combination of EDM-CDM (“and but”) are identified. The number of DM clusters and the collocation of the searched node are shown in Similarly, the collocation in the US Corpus can be obtained by employing the filter and GraphColl function. By analyzing the concordance lines with the three DMs as the nodes, 12 instances of EDM-IDM co-occurrence . The other one is that the frequency ranking of the three DMs is the same in the Chinese Corpus and the US Corpus. For instance, and appears 80 times, followed by so (45 times) and but (27 times) in the Chinese Corpus  than so  and between the US Corpus and the UK Corpus (LL = 54.48). and between corpora. However, no statistically significant difference was observed in the frequency of the DM but across the three corpora.The log-likelihood formula shows that the LL (log-likelihood) must be above 3.84 for the difference to be significant at the so, DM and. However, no statistically significant difference is found between corpora in terms of the frequency of DM but.The above pairwise comparison displays that there is a statistically significant difference between corpora regarding the use of DM “and so,” which occurs once in the Chinese Corpus and 12 times in the US Corpus. However, it never appears in the UK Corpus. As for the other identified sequencing patterns, “so but” only appears once in the Chinese Corpus, “but so” only occurs once in the US Corpus, “and but” occurs only once in both the US Corpus and the UK Corpus. The comparison of the frequency of the combinations of the three DMs can be visualized in As shown in so is quite similar across the three corpora since it has a higher frequency of appearing in the initial position followed by the medial position in all three corpora. As for the DM and, the positional distribution in the Chinese Corpus and the US Corpus is quite similar because the DM and has a higher frequency in the medial position followed by the initial position, notably in the US Corpus. However, the number of occurrences of the initial position of the DM and in the UK Corpus is, however, higher than that in the medial position. Another intriguing feature in the UK Corpus is that only the DM and appears four times in the final position. In the US Corpus, the DM but has a higher frequency in the medial position followed by the initial position, as seen in but occurs more in the initial position followed by the medial position.The present study compares the frequency of the three selected DMs, their co-occurrences, and their positions in the three corpora by adopting a corpus-based approach and a quantitative method. The results, to some extent, offer some evidence for the comparability of the three corpora.and ranks first. However, there are slight differences in the ranking order. For instance, the ranking order of the Chinese Corpus , which is demonstrated in the present study. The results of the patterns reveal a solid tendency for the combination of different types of DMs. “and so” indicates that the upcoming utterance is a result or conclusion, while “so and” marks a topic shift. The one that should be placed first or second is determined by DM functions. The DM sequencing “and so” indicates that it is frequently used to start a new topic or turn, particularly in the US corpus. In addition, “and so” is regular use in American English  and the US Corpus (25.16 vs. 74.84%) and the DM but in the US Corpus (34.78 vs. 65.25%), in which the medial proportion is greater than that of the initial position. As shown in so is less flexible than that of the other two DMs, whose initial position is always the first. This finding, however, contradicts so in the utterance medial position constitutes a greater proportion. Moreover, they rarely occur in the utterance final position. Only the DM and appears four times (4.6%) in the final position in the UK Corpus (Finally, the general positional distribution of the three DMs across the three corpora is quite similar—with the initial position having the highest proportion, followed by the medial position and the final position . This isK Corpus . Thus, wK Corpus . The DM K Corpus . Three iK Corpus . More thso occurs most frequently in the Chinese Corpus; the US Corpus has the highest frequency of the DM and; and the UK Corpus has the highest frequency of DM but, which provides a basis for a more productive analysis of the types of DMs used in media talk. Do all IDMs, for example, appear frequently in Chinese media discourse? Or are there more EDMs in American interviews? Or are CDMs more likely to occur in British media discourse? The article found that the DM cluster “and so” is most frequently used in American interviews, confirming that this combination is typical use in American English. The higher frequency of DMs appearing in the initial position also confirmed that the more interactive the genre, the more likely DMs appear in the initial position.The present study examined and compared the frequency, patterns, and positional distribution of the three DMs in the three corpora. The study concluded that there are statistically significant differences and marginal variations within and between corpora in different aspects. The study differed most significantly from previous ones in terms of the data type and objectives. While previous studies concentrated more on qualitative analysis of DM functions in academic settings or only one of the above-mentioned aspects, this study attempted to investigate all three aspects in one single goal in media discourse from a quantitative perspective. The study revealed that DM Hence, the study contributes to the existing literature by filling the gap and adding more insights into the field of discourse markers. Apart from academe, the study also sheds light on the role of DMs in interviews, which may inspire hosts and guests to consider how to achieve the best bilateral communication by using certain DMs. Despite the foregoing insights, this study falls short of expectations and does have some limitations. The findings in the present study cannot be generalized due to the small data set. Although this study provides some preliminary explanations for the differences, it fails to account for more specific variables due to the small sample size and the scope of this study. This opens new avenues for the future research in encompassing both the large corpus and another genre in order to address this problem. It is intended that the results presented in this study would be of benefit not only to individuals who conduct interviews for the media and those who are interviewed for the media, but also to those who aspire to be successful doing DM research in media settings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Both authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


AGEM = 8;0) living in the UK who have Romanian as heritage language (L1) and English as majority language (L2) and 32 Romanian monolingual children aged 6;11 to 9;8 . We examined whether differences emerge between heritage and monolingual children in the online comprehension and in the production of multiple interrogatives in Romanian, which requires fronting of all wh-phrases, contrary to English. The main aim was to uncover to which extent similarities or differences in morphosyntactic properties between the L1 and the L2 systems affect the acquisition and processing of the heritage language/L1. Online comprehension was assessed in a self-paced listening task, while production was assessed using an elicitation task. The results reveal that Romanian heritage children show similar online comprehension patterns to monolingual children for multiple interrogatives in Romanian. A different pattern emerges for production as heritage children produce less complex multiple questions in Romanian and avoid movement of two wh-phrases in all elicited structures. Given that their predominant responses for multiple interrogatives only make use of the structural option present in English, namely one fronted wh-phrase and one in-situ, we take this to show that there is transfer from the majority language to the heritage language. Thus, language production in the children’s L1 seems to be affected by properties of the dominant L2, under cross-linguistic influence. Taken together, the results for both comprehension and production suggest that heritage children are able to establish the underlying representation of multiple wh-movement structures, similarly to monolinguals, but have difficulties activating the more complex structure in production.This study compared the online comprehension and the production of multiple interrogatives in 18 Romanian-English bilingual children aged 6;0–9;2 ( Various studies on heritage language (HL) acquisition have investigated the end-state grammars of adult HL speakers ,b and hain situ. By investigating performance under different modalities, we aimed to get a more straightforward glimpse at the nature of the differences between child heritage speakers and child monolingual L1 speakers and how this relates to cross-linguistic influence , and also wh-in-situ questions as in *Jij doe wat giraffe? (lit. You do what giraffe?). According to Strik and Pérez-Leroux, complexity is a trigger for cross-linguistic influence such that structures involving less derivational complexity in one language  may impact structures which are derivationally more complex in the other language .The acquisition of wh-dependencies in the HL has also received little attention. These previous works reporting different performance patterns in heritage compared to monolingual children assessed only children’s productive skills in their heritage language/L1. Various studies with monolinguals and bilinguals have revealed asymmetries between comprehension and production  and althwho-questions and (2) exemplifies which-questions in Romanian.Full acquisition of multiple wh-questions involves various aspects that are subject to cross-linguistic variation. We will briefly outline the properties of multiple wh-questions that Romanian-speaking children need to acquire, by putting emphasis on differences with English. (1) illustrates multiple 1.a.  Cine    pe cine  acoperă?    who.Nom PE who  covers    ‘Who is covering whom?’ b.  *Pe cine  cine  acoperă?    PE who  who covers    *‘Whom is covering who?’j îlj   acoperă?2.a.  Care  fată  pe care  băiatj    himj  covers    which girl  PE which boy    ‘Which girl is covering which boy?’j care  fată  îlj   acoperă?  b.  Pe care  băiatj  which girl himj  covers    PE which  boy    ‘Which boy is which girl covering?’lexical properties of wh-words, wh-objects in Romanian are marked with a differential object marker pe, similar to a in Spanish. Although in the prescriptive use of English, who shows overt case-assignment in the form of whom, increasingly native English-speakers use who instead in informal spoken contexts (care (‘which’)-phrases in Romanian are doubled by a co-indexed clitic pronoun îl (‘him’) for masculine and o (‘her’) for feminine.In terms of contexts . Additiomovement properties, wh-words move overtly, the difference with respect to English being that multiple wh-words in Romanian move together to a clause-initial position, as shown by (1) and (2) above. This is a property that Romanian shares with Bulgarian and other Slavic languages. According to via a ‘tucking in’ mechanism , as indicated by the asterisk in example (1b). Movement of wh-words in both languages obeys Superiority  is “The girl covers the dog and the boy covers the cat.” in which the exhaustive sets of who and which are pairwise linked.The wh-questions. The acquisition of multiple wh-questions has received relatively little attention in the literature. -in-situ language. Grebenyova’s findings demonstrate that English- and Malayalam-acquiring children have adult-like knowledge of the syntax of multiple wh-questions, whereas Russian-speaking children allow fronting of only one of the wh-phrases, following an English-like structure.Children’s experience with such sentences is extremely reduced. wh-questions . wh-element present in the question. In the same vein, wh-word, which was also the subject.To our knowledge, three studies so far investigated the acquisition of multiple wh-questions in Romanian and they all looked at how Romanian-speaking monolingual children ranging in age from 4 to 9 years old comprehend this type of question. who and which-questions, who- compared to which-phrases, as measured by reaction times (RTs) in a self-paced listening task. However, only adults seem to show an online sensitivity to the ordering constraints in who-questions illustrated in (1b) above. Bentea & Marinis also report higher accuracy scores with multiple who- than which-questions and show that the latter pose more difficulties for comprehension, particularly in the object-subject order , where participants  show a preference to interpret the first wh-element as agent, along the lines of what has been reported for the processing and comprehension of simple which-questions. Bentea & Marinis also found that children even at the age of 6 and 7 answered only one of the wh-phrases, similarly to exhaust the question domain and also pair the two wh-elements. Therefore, the question that arises is whether bilinguals, who receive less input than monolinguals and are often not tutored in the L1, converge on the correct syntactic structure for multiple wh-questions and attain knowledge of the grammaticality distinctions among who and which-multiple questions, especially when such wh-dependencies display a different structure in the L2, the societal language.In a recent study on the processing of Romanian multiple To sum it up, multiple wh-interrogatives allow to explore the extent to which bilingual children’s language comprehension and production are affected by cross-linguistic influence, as they vary across languages and display language-specific syntactic and semantic properties that children need to acquire although these structures are not frequent in the parental input. In this study, we compare for the first time both the on-line/off-line comprehension and the production of these structures in Romanian heritage children in order to get a clearer picture of the way in which the societal language (here English) influences the acquisition of morphosyntax in the HL.The present study investigates the early stages in the acquisition of the HL to examine whether the differences that emerge between HL children and monolinguals hold not only for production, as has been shown by the previous studies examining the acquisition of simple wh-questions in HL children, but also for comprehension. We postulated that the use of a more sensitive and implicit on-line comprehension task, like the self-paced listening task used in this study, might offer a more straightforward glimpse into underlying language representations that are accessed for real-time processing. Together with production tasks, on-line comprehension might help to better understand what differentiates between HL children and monolingual children. The study focused on Romanian as heritage language and addressed the following research questions:1. Do Romanian HL children and Romanian monolingual children differ when processing questions with multiple wh-fronting in an on-line processing task?Previous studies with L2 children looking at real-time sentence processing report qualitatively similar processing patterns in bilinguals and monolinguals for tense , article2. Do Romanian HL children and Romanian monolingual children differ with respect to the production of interrogatives with multiple wh-movement and how does this compare to comprehension?in-situ, under cross-linguistic from English, the majority language. Moreover, if asymmetries arise between production and comprehension, then we expect the results to show a similar pattern to that reported for other bilingual populations in which comprehension of multiple interrogatives outpaces their use in production  living in the United Kingdom (Greater London area and South-East England) and 30 Romanian monolingual children aged six to nine  living in Romania, participated in the studyEighteen 6- to 9-year-old Romanian heritage children with English as L2  and the mean length of exposure (LoE) to English was 6;0 years . The language background data for the heritage children illustrated in t-test , whereas English is the dominant language outside the home , as well as when it comes to children’s current expressive language skills, as reported by the parents .Details regarding the bilingual children’s language history, including information about their current use of and exposure to both Romanian and English, were collected using a modified version of the Questionnaire for Parents of Bilingual Children PABIQ; . Three pChildren’s ability to comprehend Romanian multiple wh-questions in real-time was assessed with an on-line SPL task with picture verification see . In this-phrases in which we crossed two factors: the order of the wh-constituents [either the wh-subject preceded the wh-object (SO) or vice-versa (OS)] and the type of wh-phrase (who vs. which). There were 40 test items in total, with 10 items per condition. The sentences included stories about superheroes  and princesses  engaging in imaginary activities with different people or animals. At the beginning of every trial, children listened to a lead-in sentence, introducing the characters. This was then followed by the test sentence  segmented into 8 parts, each part containing one to three words. No images appeared on the screen while children listened to the lead-in and the test sentence. The slashes in the examples signal the end of each segment when children had to press the Space bar to hear the next segment. After the final segment of each test sentence, a picture with three pairs of characters appeared on the screen. All the visual stimuli used in the study can be provided upon request. The position of the pairs and the direction of the actions varied between pictures. Children had to verbally identify all the pairs of characters performing the action described in the sentence. An answer was coded as “correct” when all the relevant pairs were identified. After hearing the sentence in (3), for example, the expected answer was The fireman is splashing Superman and the elephant is splashing Batman. This is because Romanian only allows exhaustive pair-list interpretations for multiple wh-questions like the ones exemplified in (3) to (6) below.The self-paced listening task in the current study was presented as a computer game with a puppet (Paddington the Bear). The children were told that they have to listen very carefully to Paddington’s questions in order to be able to identify the correct characters in the picture that followed each question. The experimental items contained embedded questions with two fronted whLead-in:    This is an image of Spiderman, Superman, Batman, a boy, a fireman and an elephant.Test sentence: Paddington/ wants to know/SO who  cine/pe cine/ stropește/în joacă/seara/           la circ/ 3.         who PE whos plashes jokingly in the evening        at the circus        “who is splashing whom jokingly at the circus in the evening.”OS who  *pe cine/ cine/ stropește/în joacă/  seara/          la circ/ 4.         PE who   who splashes  jokingly    in the evening       at the circus        “who whom splashing jokingly at the circus in the evening.”Lead-in    This is an image of Anna, Elsa, Jasmine, and three monkeys.Test sentence: Paddington/ wants to know/SO which  care  prințesă/pe care  maimuță/  o/  acoperă/  dimineața/      la zoo/ 5.         which princess  PE which monkey      her   covers    in the morning    at the zoo        “which princess is covering which monkey at the zoo in the morning.”OS which  pe care prințesă/care    maimuță/  o/  acoperă/  dimineața/      la zoo/ 6.         PE which princesswhich monkey        her  covers     in the morning    at the zoo        “which princess is which monkey covering at the zoo in the morning.”who-questions and the other half were simple subject which-questions. They included both transitive actions  in which the agent was always animate and the patient inanimate, as well as intransitive actions .Apart from the 40 test items, 10 fillers were also included as distractors. Half of the fillers were simple subject who-questions with a SO order (7), 12 items elicited multiple which-questions (six with a SO order as in (8) and six with an OS order as in (9)), while six more items were designed to elicit multiple wh-questions with an OS order (10), in which the object was a which-phrase and the subject a who-phrase, a grammatical option in Romanian.For the elicited production task, children played a guessing game with Paddington the Bear in which they were prompted to produce 24 multiple wh-questions with a subject-object and an object-subject order. Six items were designed to elicit multiple SO who     Cine  pe   cine  a  mângâiat?7.          who   PE   who  has patted          “Who patted whom?”SO which     Care  fată  pe   care  pisică  a   mângâiat-o?8.          which  girl  PE   which  cat    has  patted-her          “Which girl patted which cat?”OS which     Pe  care   pisică  care  fată   a    mângâiat-o?9.          PE  which   cat    which  girl   has   patted-her          “Which cat did which girl pat?”OS which-who  Pe  care  pisică  cine   a    mângâiat-o?10.          PE   which  cat     who   has   patted-her          “Who patted which cat?”Children interacted with a Paddington the Bear puppet for the whole duration of the task (see details in the Procedure section below).Here are two girls, a boy, two cats and a monkey, which helped them to familiarise themselves with the characters in the image. The experimenter then covered Paddington’s eyes and ears so that the puppet could not see or hear anymore what was happening next in the image. At this point, the child saw another image and heard Look! This girl is patting the black cat and this girl is patting the white cat. The boy is taking a picture of the monkey. Then the image presented at the beginning of the trial appeared again on the screen. Paddington’s eyes and ears were uncovered and afterwards the experimenter would tell the puppet Paddington, we can tell you that the boy did not pat anyone, but each girl patted a different cat. The experimenter then prompted the child to ask Paddington a question about this  and then Paddington made his guess. In order to make sure that children produced questions with two wh-phrases, they were told that they need to ask questions about two things at the same time. The task also included six filler items. These prompted children to produce simple wh-questions (three argument questions with a mismatch in animacy like “What did the father wash? and three adjunct questions such as Where did the queen sit?). Children’s responses were coded as “felicitous” when they produced a target question with multiple wh-movement like in examples (7) to (10) above. Other responses were coded as “infelicitous,” alongside the type of error produced.The structure of each scenario was as follows. First, both the child and Paddington saw an image of Lego figures and heard The study was approved by the Ethics Committee of the University of Reading. Informed parental consent was obtained for each child prior to the testing sessions, as well as the oral consent of the child. Each participant was tested individually in a quiet room either at their school or in their home. The experimenter gave oral instructions for both tasks. These were administered at least 1 week apart and in different orders, such that half of the children first saw the SPL task, followed by the elicited production task, while for the other half we first assessed production and then comprehension. Each testing session lasted around 30–40 min, but children could take breaks whenever they felt tired. They received stickers and certificates of participation after each task and at the end of the study they received as well a voucher which they could use to buy books at local bookstores.what-questions  and two multiple wh-questions with an animate subject and an inanimate object . Each participant then listened to a total of 50 sentences during the test phase and these were randomized in PsychoPy. The task instructions and all the sentences were pre-recorded by a native speaker of Romanian.The SPL task was programmed and administered using PsychoPy . The tasSO who questions) and the order of the blocks was randomized across four lists, each containing 30 items. The task began with four practice trials which elicited multiple wh-questions with a mismatch in animacy . The audio presentation of the items was also pre-recorded by a native speaker of Romanian. The questions that the children produced were recorded on answer sheets and then coded for analysis.The elicited production task was administered as a PowerPoint presentation. The task started by introducing each child to the Paddington puppet. The experimenter explained that Paddington wanted to become a magician and for this he had to improve his guessing skills. The child’s task was to help Paddington by playing a game with him in which the child asked Paddington questions about various images and the puppet had to guess the correct answer. For each correct guess, the child gave Paddington a smiley face sticker and if Paddington had at least 20 correct guesses by the end of the task, the child gave Paddington his Magician Diploma. Given the complexity of the target questions, these were presented in four blocks and interspersed with fillers. Each block contained 6 items of the same type , but show lower accuracy for multiple which-questions.The fireman is splashing Superman, the elephant is splashing Batman, and the boy is pulling Spiderman although the question only refers to the action of splashing. List answers are cases in which children only answer one of the two wh-words by exhaustively listing all the characters involved in the action. That is, they would answer question (5) with Jasmine and Elsa. Role reversals are answers in which the Agent-Patient roles are reversed. For instance, children would answer the question in (6) with Anna is covering the black monkey and thus assign the wrong thematic role to which princess. There were also very few instances in which children simply identified the wrong action, for example the pulling action when the question was about splashing. who-questions are providing list answers to only one of the wh-words, while role reversal errors are the most frequent with multiple which-questions. This suggests that children find it harder to assign the correct thematic roles in a patient-before-agent structure when the question contains two which-phrases.The two groups also show similarities in the errors they produce, which are (i) over-exhaustive answers, (ii) list answers, and (iii) reversed role answers. For over-exhaustive answers children name all the pairs in the image, even when one pair performs a different action. For example, children would answer a question like in (3) or (4) above with lme4 package (emmeans package (Given the binary nature of the data (Correct/Incorrect), we analyzed the accuracy results using a binomial generalized linear mixed model with group , wh-type (who vs. which), wh-order (SO vs. OS), and their interaction as fixed factors. The fixed factors were coded using repeated contrast coding which tests the difference between the mean of the dependent variable for one level of the categorical variable and the mean of the dependent variable for the adjacent level . The ran package  in R (R  package . Planned package .p = 0.988). The summary of the final model is given in who-questions were comprehended better than which-questions) and a significant wh-type*wh-order interaction. No differences in performance emerged between the monolingual and the heritage group , while there was a significant difference between the two wh-orders in the case of questions with two which-words, with SO which questions yielding significantly better comprehension scores than OS which questions .We followed-up on the significant interaction between wh-type and wh-order with a pair-wise comparison. This showed that response accuracy for questions with two online processing of multiple wh-questions are illustrated in SD above or below the mean per condition per participant and per item were considered as outliers and therefore replaced with the mean per condition per participant and per item. The total proportion of extreme values and outliers was 3.2% of all data points.The segment-by-segment residual reaction times (RTs) for the p were calculated by way of Satterthwaites’s approximation to degrees of freedom with the lmerTest package , wh-type (who vs. which), wh-order (SO vs. OS), as well as their interaction, were included in the fixed effects structure for each model. All fixed effects were coded using repeated contrast coding. Values of  package .Segment 1 (the first wh-word) Results attested to a significant effect of group , indicating that the monolingual children had overall longer RTs than the heritage children. There was also a significant effect of wh-type , with longer RTs for who-phrases (M = 482 ms) than which-phrases (317 ms), as well as a significant effect of wh-order , with shorter RTs for subject (M = 371 ms) compared to object wh-words (458 ms). None of the interactions was significant.Segment 2 (the second wh-word) Results revealed a significant effect of group , with the monolingual children displaying longer RTs than the heritage children, and a significant effect of wh-type , with longer RTs for who-phrases (M = 484 ms) than which-phrases (352 ms). No other effect or interaction were significant.Segment 3 (the verb in multiple who-questions and the clitic in multiple which-questions) There was a significant effect of group , with longer RTs for the monolingual group compared to the bilingual group, and a significant effect of wh-type  showing that the verb segment in the multiple who-conditions yielded shorter RTs (M = 386 ms) than the clitic segment in multiple which-conditions (M = 638). There was also a significant interaction Group*WhType . As a follow-up on the significant interaction, pair-wise comparisons revealed that the heritage group showed significantly shorter RTs than the monolingual group for the who-conditions, , while no significant differences surfaced between the two groups in the which-conditions.Segment 4 (the adverb in multiple who-questions and the verb in multiple which-questions) No effect was significant.Segment 5 (in the morning) Results attested to a significant effect of group , as the monolingual children had longer RTs than the heritage children. No other effect was significant.Segment 6 (at the zoo) No effect was significant.To summarize, the results of the comprehension task show similar response accuracy and a similar pattern during online comprehension of multiple wh-questions in Romanian-speaking monolingual and heritage Romanian children.The results of the elicitation task showed that children do not only produce questions with multiple wh-fronting, the expected target structure based on the syntax of Romanian multiple wh-questions, but that they often produce other structures as well. To reflect this variability in the children’s answers, four scoring categories were used, each corresponding to the four main question types that children produced and which we classified as follows:who or which, and with both wh-words fronted, as in (11). a. MWH_MULTIPLEMOVE: when children produced a question with two wh-words, either Cine pe cine   a mângâiat ?11.   who PE whom has patted  “Who patted whom?”b. MWH_SINGLEMOVE: when children produced a question containing two wh-words in which only one wh-phrase is fronted and the other one appears in-situ, like in (12). Cine a mângâiat pe cine ?12.   who has patted PE whom  “Who patted whom?”c. SIMPLE_WH: when participants produced a grammatically correct question but only with one fronted wh-word (13). Care  fată a mângâiat pisica?13. F.SG  which girl  has patted  cat.the.  “Which girl patted the cat?”d. COORDINATED_WH: when children produced a question with two coordinated wh-words (14). Cine și  pe cine a mângâiat ?14.   who and PE who has patted?  “Who and whom is punching?”The distribution of responses differs between the Romanian monolingual and Romanian heritage children, as can been seen in SO which and OS which questions and thus report the data for three types of multiple wh-questions with two who-phrases (who), with two which-phrases (which), and with one which and one who phrase (which-who). in-situ) represent the preferred produced structure for the heritage group across all types of elicited questions, irrespective of whether these contained only who, only which, or both which and who phrases. The monolingual group produce more MWH_SINGLEMOVE structures of the type illustrated in (12) with questions containing which-phrases, whereas they produce more MWH_MULTIPLEMOVE structures like in (10) in the presence of two who-elements.A finer characterization of the results  gives anwho vs. which vs. which-who), we fitted a generalized liner mixed model for each of the four scoring categories outlined above, namely MWH_MULTIPLEMOVE, MWH_SINGLEMOVE, SIMPLE_WH, COORDINATED_WH. The dependent variable in each model was response accuracy, that is, the correct production of questions within each scoring category. We analyzed the production data using the lme4 package (who vs. which vs. which-who) as well as their interaction. We used a repeated contrast specification for the fixed factors. The random effect structure included by-participant random slopes. We only report the significant effects and interactions.In order to uncover whether differences emerge between monolingual and heritage children in their productions and whether these differences are modulated by the type of elicited question . Overall, the heritage children produced significantly fewer questions with multiple wh-fronting compared to the monolingual children. The analysis also revealed a significant difference between which and who questions . Multiple wh-fronting was most frequent when children had to produce questions with two who-phrases compared to two which-phrases. The interaction Groupvs MonolingualHeritage *QuestionTypevs whichwhich-who  was also significant . As a follow-up on the significant interaction, pair-wise comparisons with an adjusted alpha level using the Tukey method showed that the monolingual group produced significantly fewer questions with multiple wh-movement when the target items only contained which-phrases compared to when they contained one who-phrase and one which-phrase .The statistical analysis revealed a main effect of Group . This means that, overall, heritage children produced significantly more multiple wh-questions with one element fronted and one in-situ than the monolingual children. There was also a significant difference between which and who questions , indicating that there were overall more multiple wh-questions with single wh-fronting for which-questions relative to who-questions.The statistical analysis revealed a main effect of Group . This means that children produced less coordinated wh-questions when both wh-words elements were which-phrases. There was also a significant Groupvs MonolingualHeritage *QuestionTypevs whowhich  interaction . Subsequent pairwise comparisons show that the heritage group produces significantly fewer coordinated wh-questions when the target items contained two which-phrases compared to when these contained two who-phrases  and this difference is more pronounced in heritage children than in monolinguals.The statistical analysis revealed a significant difference between in-situ compared to the monolingual children.Thus, heritage children display a different pattern than monolinguals in production, as they produce significantly fewer questions with multiple wh-fronting and significantly more questions with one fronted wh-phrase and one who- and which-questions. The on-line task investigated how Romanian heritage children and Romanian monolingual children process wh-dependencies with two fronted wh-words, while they were listening to sentences on-line for comprehension. This task also aimed to find out whether Romanian-speaking children were sensitive to the asymmetry in object-over-subject movement between multiple who- and which-questions in real-time comprehension. The combination of production and on-line comprehension tasks can help to get a better picture of heritage language development and better understand the relationship between performance in production and real-time processing.The main questions guiding the present study were whether differences emerge between Romanian-English bilingual children, for whom Romanian is the heritage language, and Romanian monolingual children in the on-line comprehension and production of multiple wh-interrogatives and whether these differences are due to cross-linguistic influence from the dominant English. To address these questions, we used an on-line comprehension task and an elicitation task targeting multiple who-subject and who-object well, irrespective of the order in which the two wh-phrases occur. In contrast, they have more difficulties with the comprehension of multiple which-question, particularly when the wh-object precedes the wh-subject. This is in line with cross-linguistic findings reported for the comprehension of simple which-questions showing that children find object which-questions harder to comprehend than subject which-questions and that the type of wh-element also affects comprehension of wh-dependencies, with object who-questions being acquired earlier thn object which-questions , but also questions in which only one wh-phrase is fronted, the other one remaining in-situ. This option exploited in production surfaces mostly in questions with two which-phrases. In addition, monolingual children also produce a significant number of simple wh-questions, that is, questions with only one wh-word. While heritage children also produce simple questions, they produce significantly more questions with one fronted wh-phrase, one in-situ than monolingual children and, with the exception of one child, avoid multiple wh-movement, contrary to monolinguals. Importantly, there were no instances of multiple who-questions with an ungrammatical object-subject order in any of the children’s productions. This indicates that their grammatical system does not allow this option and further reinforces the idea that their lack of sensitivity to the object-over-subject ungrammaticality in multiple who-questions in on-line comprehension is not due to a different grammar than that of adults, but it most likely stems from the processing load associated with such complex structures involving multiple movement dependencies.Let us now turn to the elicitation task which examined whether Romanian heritage and monolingual children are able to produce questions with multiple wh-movement and whether they have fully acquired the specific syntax of multiple interrogatives in Romanian, which requires fronting of all wh-phrases. Contrary to comprehension, we found differences in the production of multiple wh-questions between Romanian heritage and monolingual children. Monolingual children produce questions with multiple wh-fronting  are acquired earlier and also that they are more likely to influence structures which require more complex derivations (like multiple wh-fronting), in line with a complexity-based theory of transfer , indicating that they treat multiple wh-questions as simple questions. These errors in comprehension, together with children’s production patterns, show that even monolingual Romanian-speaking children take longer to produce structures with multiple wh-fronting and that they also make use of the option of having one wh-fronted and one in-situ. This corroborates An interesting observation here is the fact that children’s production patterns mirror the errors that they produce in the comprehension task. For example, reversed role errors are linked to children’s preference to mainly produce questions with a subject-object order for the conditions containing Our study makes a substantial contribution to the understanding of child HL development, as it investigates the production and comprehension of the same phenomenon in HL speakers and provides insight into the language development stages of both monolingual and heritage bilingual children. The current study is among the first to investigate cross-linguistic influence in bilingual children during real-time sentence processing and the first to use the self-paced listening paradigm with heritage children. However, the study also paves the way to future questions that remain unaddressed, such as when the syntax of multiple interrogatives is fully acquired as well what happens with adult heritage speakers or child heritage speakers whose dominant language(s) also have multiple wh-movement or lack this type of questions entirely. Studies that compare multiple bilingual groups will further increase our understanding of the impact of cross-linguistic influence on heritage language development.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Reading. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AB carried out the data collection and was responsible for data analysis. AB and TM shared responsibility for the conception of the work and the interpretation of results. AB wrote the manuscript with input from TM. All authors contributed to the article and approved the submitted version.This research was supported by an Early Postdoc. Mobility grant for AB from the Swiss National Science Foundation (grant number 174870).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The role of the pulmonary microbiome in sarcoidosis is unknown. The objectives of this study were the following: (1) examine whether the pulmonary fungal and bacterial microbiota differed in patients with sarcoidosis compared with controls; (2) examine whether there was an association between the microbiota and levels of the antimicrobial peptides (AMPs) in protected bronchoalveolar lavage (PBAL).Thirty-five sarcoidosis patients and 35 healthy controls underwent bronchoscopy and were sampled with oral wash (OW), protected BAL (PBAL), and left protected sterile brushes (LPSB). The fungal ITS1 region and the V3V4 region of the bacterial 16S rRNA gene were sequenced. Bioinformatic analyses were performed with QIIME 2. The AMPs secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 and hBD-2), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA).Aspergillus dominated the PBAL samples in sarcoidosis. Differences in bacterial taxonomy were minor. There was no significant difference in fungal alpha diversity between sarcoidosis and controls, but the bacterial alpha diversity in sarcoidosis was significantly lower in OW (p = 0.047) and PBAL (p = 0.03) compared with controls. The beta diversity for sarcoidosis compared with controls differed for both fungi and bacteria. AMP levels were significantly lower in sarcoidosis compared to controls (SLPI and hBD-1: p < 0.01). No significant correlations were found between alpha diversity and AMPs.The pulmonary fungal and bacterial microbiota in sarcoidosis differed from in controls. Lower antimicrobial peptides levels were seen in sarcoidosis, indicating an interaction between the microbiota and the innate immune system. Whether this dysbiosis represents a pathogenic mechanism in sarcoidosis needs to be confirmed in experimental studies.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-022-01362-4. Sarcoidosis is a systemic inflammatory disease that can be challenging to diagnose and treat. Disease manifestations are heterogeneous, with symptoms varying considerably . The disMycobacterium and Propionibacterium has been widely studied [The causative agents inducing the immune response in sarcoidosis are yet somewhat of an enigma, but the immune system seems to respond to different substances by producing inflammatory cells organized in granulomas. It has long been recognized that the immune response could reflect an infectious etiology and scientists have for decades searched for that one pathogen that might cause sarcoidosis , 2. Desp studied .Aspergillus [Novel culture independent techniques as high-throughput 16S rRNA gene sequencing has revealed the presence of a diverse bacterial microbiota in the lungs . In sarcergillus , 6. A smergillus , 7, 8 orergillus –12. The ergillus , 12 and ergillus , 10.Antimicrobial peptides and proteins (AMPs) are key effector molecules in the innate immune system against respiratory pathogens , 14. TheThe aim of this bronchoscopy study was to examine whether the fungal and bacterial pulmonary microbiota differed in patients with sarcoidosis compared to healthy controls, and if there was an association between the microbiota and the AMPs.runiform  function in Stata 14.2 [Participants were recruited from our outpatient clinic at the Department of Thoracic Medicine, Haukeland University Hospital, Norway, participating in The Microbiome in Interstitial Lung Disease study (MicroILD)  and The ata 14.2 . MetadatThe participants underwent bronchoscopy with oral access and topical anesthesia in a supine position. Intravenous alfentanil (0.25–1.0 mg) was offered. To minimize contamination, no suctioning was performed above the vocal cords. During bronchoscopy we collected 3 protected sterile brushes from the left upper lobe (LPSB) and 3 × 50 ml (2 × 50 ml for controls) of bronchoalveolar lavage from the right middle lobe through a sterile protected sheath (PBAL). Sterile phosphate-buffered saline (PBS) was used for all fluid samples, including 10 ml of oral wash (OW). A negative control sample was obtained from the PBS bottle used for the procedural samples of each participant, without being exposed to the participant or the bronchoscope.The 16S rRNA sequencing was performed during the course of the MicroCOPD and MicroILD studies and included sequencing of more than 2500 samples from almost 400 bronchoscopies, with up to 8 sampling sites per participant. For the ITS sequencing which was performed afterwards, we chose to limit the sequencing to PBAL, OW, and negative control samples, due to cost restraints.The detailed protocol DNA extraction, PCR and sample preparation for MiSeq sequencing for the 16S rRNA gene analyses is previously published . The AddThe bioinformatic analyses were performed with QIIME 2 .Cytospin slides were made from PBAL and stained with May-Grünwald-Giemsa. A minimum of 300 cells were counted for differential cell counts by an observer blinded for the patient’s data. The AMPs, SLPI , and hBDFor statistical analyses we used Stata  and QIIMThe demographics of the participants are shown in Table Saccharomycetes was the most abundant fungal taxa in the OW samples for both study groups and PBAL samples for controls. However, in PBAL samples for sarcoidosis patients, the taxa were more evenly distributed, with the most frequent classes being Eurotiomycetes and Malasseziomycetes. At genus level, Candida was most abundant in the OW samples for both categories, while being less abundant in PBAL samples for patients compared to controls. Aspergillus was the most frequent genus measured in PBAL samples for sarcoidosis patients but was undetectable in PBAL and OW samples for controls. ANCOM analysis at class level showed significantly less Saccharomycetes in PBAL samples and less Sordariomycetes in OW samples from sarcoidosis patients compared to controls , there were significantly less Abscondibacteria in PBAL and Fusobacteria in OW for sarcoidosis patients compared to controls at phylum level, and significantly more Stomatobacteria in OW and Synergistia in LPSB in patients compared to controls at genus level  and Bray-Curtis (p = 0.03), and in OW by Jaccard (p = 0.04) , in PBAL by unweighted UniFrac (p = 0.02); and in LPSB by weighted UniFrac (p = 0.02), unweighted UniFrac (p = 0.03), Bray-Curtis (p < 0.01), and Jaccard (p < 0.01)  and hBD-1 (p < 0.01) (Table The levels of AMPs in the PBAL samples were significantly lower in sarcoidosis compared to controls for SLPI p < 0.0 and hBD-1) Table .This study demonstrated that stable sarcoidosis patients have a different fungal and bacterial microbial composition in the lower airways and oral cavity compared with healthy controls, being most pronounced for fungi. Differences between sarcoidosis and controls were found when examining differential abundances of taxonomy and differences in microbial diversity. The lower airways levels of the antimicrobial peptides SLPI and hBD-1 also differed between sarcoidosis and controls, with lower levels in sarcoidosis. However, we did not find a statistically significant association between the alpha diversity and the examined antimicrobial molecules.Aspergillus nidulans when examining BAL fluid in 9 patients with Lofgren syndrome, compared with 3 controls, together with increased serum antibodies, and the authors suggested that Aspergillus nidulans had a potential pathogenic role [Aspergillus  in BAL fluid but questioned this to be contamination [Fusobacterium spp and Atopobium spp in sarcoidosis compared to healthy controls [Erythrobacteraceae family in sarcoidosis patients [Actinobacteria and Proteobacteria to be significantly more abundant in sarcoidosis patients compared to ILD patients [Pneumocystis pneumonia patients and 9 healthy controls [Since the airways are not sterile in healthy individuals, a potential hypothesis is that a dysbiosis of the lower airways microbiota is a causal factor in the aberrant immune response seen in several chronic lung diseases. The role of the microbiome in the pathogenesis of pulmonal sarcoidosis has only been examined in a few studies, without consistent results. A recently published paper by Greaves et al. found enrichment of a peptide from nic role . Clarke mination . In anotcontrols . Fukui epatients . Gupta epatients . Three opatients , and a scontrols . All thrCandida was the most abundant genus in the OW samples for both groups and in the PBAL samples from healthy controls. Candida was significantly lower in PBAL samples for sarcoidosis patients compared to controls, possibly driven by Aspergillus, which was the most frequent taxon in this group. This was in line with the two other known studies on the fungal pulmonary microbiota in sarcoidosis patients as previously described [Candida and Aspergillus are the most prevalent and well-known fungal pathogens [ladosporium and Penicillium [In our study, the differences between patients and controls were more striking for the fungal taxonomy compared with the bacterial taxonomy. escribed , 6. Candathogens . Increasathogens . Antifunathogens , but neeicillium , 26, whiFirmicutes and Bacteroidetes, while Streptococcus, Prevotella, and Veillonella dominated the genus level, all shared by both study groups. These taxa are described as the most common bacteria in both the lower and upper airways in a healthy lung [The most abundant bacterial phyla we detected were thy lung . DiffereAntimicrobial peptides are produced by immune and inflammatory cells in the lungs and other mucosal tissues, and exhibit broad spectrum antimicrobial, immune modulatory, and wound repair features . These mInterestingly, we found significantly lower values for SLPI and hBD-1 in our study for sarcoidosis patients compared to controls. Agerberth et al.  examinedSince our study is cross-sectional, we cannot differentiate between lower levels of anti-microbial peptides leading to a microbial dysbiosis, or vice versa. Low levels of antimicrobial peptides in the airways could be a pathogenic factor, or a sign of an overwhelmed innate immune response. In any event, the persistence of granuloma and inflammation in sarcoidosis point to activation of stimulating antigens driving the disease process, and our study indicates that the fungal microbiota may be part of this process.Accurately measuring the low biomass microbiota in the lower airways is challenging. The bacterial DNA density in the upper airways is at least 100-fold higher than in the lower airways . Low bioHowever, our study has some shortcomings. First, BAL yield will differ within both the patients and control groups, which could influence microbial composition and measured levels of the AMPs. Mean BAL yield was 47.2% (SD 11.6) for controls and 48.3% (SD 17.7) for the patients, thus reasonably similar but with larger spread for the patients. Second, our study lacks patients with acute sarcoidosis in the early stage of disease. Third, fungi are even more delicate to examine with next-generation sequencing compared to bacteria due to its low abundance in many samples, high levels of fungal DNA from contamination sources and finally lack of standardization in primer designs, reference databases, and analytic methods , 37. TheAspergillus genera dominated the lower airways in sarcoidosis. In addition, sarcoidosis patients had lower levels of antimicrobial peptides in the airways. These findings could indicate the presence of a microbial dysbiosis in the airways in sarcoidosis. Future research should address whether this dysbiosis has a pathogenic role, and thus be a potential target for new treatment principles.In this study, we found differences in fungal and bacterial diversity between sarcoidosis patients and controls, and a more clearly distinct fungal taxonomy in the lower airways compared with controls, where Additional file 1. Supplementary Methods.Additional file 2: Supplementary Table 1. Comparison of beta diversity by pairwise PERMANOVA between the sarcoidosis patients and controls for fungal and bacterial taxa sampled by different methods* and assessed by different beta diversity metrices. Supplementary Table 2. Spearman's correlation coefficients (rho) and p-values for differences in alpha diversity correlated with antimicrobial peptides.