While task-based peer interaction in dyads has been commonly practiced in English as a foreign language (EFL) classrooms, how to pair learners in dyadic tasks has always been a concern of teachers and researchers. This study examined learner proficiency pairing, task type and L1 use by Chinese EFL learners in two dyadic speaking tasks. Thirty-six participants were paired according to their oral English proficiency levels into: same-level pairs , and mixed-level pairs (high-low). All pairs completed two types of speaking tasks—information-gap and opinion-exchange. Quantitative results showed a significant difference between low-low pairs and other pairs in the amount of L1 use. Low-level learners produced significantly more L1 words and turns when paired with other low-level peers (low-low) than with high-level peers (high-low) in both types of tasks. Qualitative analysis further indicated that the mixed-level (high-low) pairs produced more opportunities for negotiation of meaning than the same-level (low-low) pairs during the interactional episodes where the L1 served various functions. The study offers pedagogical implications for EFL teachers about how to optimally pair learners to maximize their language development. Second language (L2) interactionists have long claimed that conversational interaction between non-native speakers through communicative tasks can enhance speaking opportunities  focusing on two types of speaking tasks, which were common practices in EFL university classrooms with large class sizes; (2) adopting a counter-balanced task design so as to eliminate the potential impact of task sequence on task performance; (3) administering an oral English proficiency test to accurately measure learners' L2 proficiency levels before pairing; (4) diversifying the L2 proficiency pairings used in previous studies by incorporating medium-medium (M-M) learners. In addition to the type and frequency of L1 functions, this study also delved into whether these L1 functions affected opportunities for negotiation of meaning through interactional moves in those episodes where L1 functions occurred, so as to gain a full picture of the functions served by L1 use. The following research questions were then examined:The study was conducted in a top-tier university in Beijing, China, where English is a compulsory subject for first-year students. These students have learned English for about 9 to 12 years. They were non-English major students, majoring in varieties of subjects in social science see . They reAn invitation to participate in our study was sent to students in the three English Listening and Speaking classes taught by the first author, and fifty-two students initially agreed to participate. An oral proficiency interview was then administered to these students to measure their English oral proficiency level. Forty students were finally selected as the target participants due to the study's proficiency pairing needs. They were then paired into four different types of proficiency pairing based on their English oral proficiency levels: same-level pairs , and mixed-level pairs (H-L), with five pairs for each pairing type. However, due to two L-L pairs dropping out in the second round of data collection, only the interaction data of the 36 students who attended both data collection sessions were analyzed. These 36 students, with an average age of 19, were randomly paired, disregarding gender and subject backgrounds see .An interview was conducted by the two authors to rate the participants' oral English proficiency levels. It included two parts: self-introduction and a paired oral task. The rubric of the interview followed the five scales of International English Language Testing System (IELTS) proficiency guidelines: fluency and coherence, pronunciation, grammatical range and accuracy, lexical resources, and discourse management. The two interviewers scored each student separately and an agreement on the proficiency level of the participants was then reached with an inter-rater reliability of 0.94. For the convenience of pairing, participants' English oral proficiency levels were labeled roughly as low, medium, or high.This study examined two types of speaking task: an information-gap task and an opinion-exchange task see . AccordiDifferent from the collaborative writing tasks used in previous studies , whereas percentages of L1 words  and L1 turns  were not. The two authors conducted all the L1 counting and coding, and agreement was reached for any inconsistencies.The audio recordings of participants' oral interactions were first transcribed verbatim. The amount of Chinese characters and English words was then counted for each participant and in each task, together with the amount and frequency of L1 turns. The quantitative data on the percentage of language use was entered into SPSS for statistical analysis. Descriptive statistics were first extracted to gain their mean and standard deviation. Inferential analysis was then implemented for between-group and within-group differences. The Kolmogorov-Smirnov test was used to examine the normality of data distribution before executing parametric (ANOVA) or non-parametric tests . Total words were normally distributed  and opinion-exchange tasks . Wilcoxon's signed ranks test revealed no significant difference between the L1 percentages of these two task types  for all proficiency pairings  and opinion-exchange tasks . The low-level students in the L-L and H-L pairs seemed to produce L1 turns in different ways. On average, the low-level students in L-L pairs produced far more L1 turns than those in H-L pairs in both tasks.The abovementioned quantitative findings indicated a significant difference in the amount of L1 words and turns between H-L and L-L pairs. The L1 functions of these two types of pairings were mainly differentiated in relation to metacognitive and vocabulary talk. We examined the interactional moves in the task-related interactional episodes where L1 and L2 use served the same functions , with regard to the opportunities for negotiation of meaning which might be elicited by these moves.We examined the interactional moves in the task-related interactional episodes where L1 and L2 use served the same metacognitive talk functions, with regard to the opportunities for negotiation of meaning which might be elicited by these moves. The following examples (1) and (2), produced by L-L (Pan-Rui) and H-L (Huang-Ting) pairs during the same opinion-exchange task on the effects of English mania, are good cases in point.Example (1)Pan (L): Hello, everyone, today I'm talking about the mania of English.Rui (L): So what's your view?I should say it by myself), , (I say it by myself). I hold a positive view about the phenomena, because I think English helps …… Another reason is that English allows …Pan: (Example (2)Huang (H): I hold a positive view about the English mania. How about you?Ting (L): I hold a positive view. So our team holds a positive view?Huang: It is too early to make a conclusion. So what is your reason?Ting: The reason?Huang: A better future we can have and a better job for high salary. Our ability to speak English, you can see here, because many well-pay jobs require knowledge of English.Ting: Yeah.Huang: And learning of English will surely have a positive effect on any of us who study English as a second language. This is the first reason. That is to say, better life and better job.Ting: I think the second reason is … (pauses here for 3 seconds) English can help …err (pauses here for 2 s) help us to learn foreign country, and people and culture.The episodes above revealed different interactional moves and opportunities for negotiation of meaning. In the L-L pair (Example 1), Pan initiated the conversation in turn 1, with the intention of stating her viewpoint and pretending that she was giving a presentation on her own in class, which was her understanding of the task. When Rui interrupted her by requesting clarification in turn 2, Pan became anxious in turn 3 and used L1 to inform him of her understanding of how the task should be completed. Pan used L1 as a strategy to immediately bring the conversation back on track in order to move the task along. However, this quick resolution of communication difficulty over task management effectively shut down Rui's opportunity to negotiate.However, the H-L pair in Example (2) demonstrated completely different interactional features when a disagreement on completing the same task arose. In turn 2, Ting (L) requested clarification of their view, which was obviously too early to do so according to the teacher's task instructions. In turn 3, Huang (H) explained this to Ting and guided her to state her reasons. When Ting again requested clarification in turn 4, Huang set an example for her in turn 5. As Huang continued to elaborate more on her view in turn 7, Ting seemed inspired and voluntarily took the turn to join the conversation in turn 8, which could be deemed as successful pushed output. Although the same communication difficulty in task management was resolved more slowly in this example compared with Example (1), with the assistance of Huang, the high-level peer, further clarification requests generated more opportunities for both learners to negotiate meanings in L2.It can be seen that, compared with L-L pairs who tended to use L1 as pedagogical strategies, the H-L pairs generated more interactional moves for metacognitive talk and opportunities for negotiation of meanings in L2.fatigue.The same goes to the interactional moves of vocabulary talk. When completing an information-gap task about a university student consulting fitness club programs, the L-L pair in Example (3) and H-L pair in Example (4) encountered the same lexical difficulty of understanding the word Xiong in the L-L pair requested clarification in turn 2 to indicate a linguistic difficulty, and Wang repeated the L2 word and offered an L1 equivalent immediately afterwards in turn 3 before continuing talking. However, when receiving Shan's (L) clarification request in turn 2, Sui (H) in the H-L pair repeated the L2 phrase and offered an L2 explanation in turn 3. Shan immediately followed this with a confirmation check by repeating the L2 synonym with a rising intonation in turn 4, which was confirmed by affirmative feedback from Sui in turn 5. Again, although the same lexical difficulty was resolved more slowly than in Example (3), with the assistance of Sui, the high-level peer, the clarification request generated a confirmation check that allowed more opportunities for the low-level learner to produce the L2 synonym herself, thus extending her focus on and understanding of the meaning of the unknown word.Example (3)Wang (L): It can reduce body fat, keep fatigue away.Xiong (L): keep what away?fatigue), can help you manage stress …Wang: fatigue, (Example (4)Sui (H): And in the mental level, it can keep fatigue away.Shan (L): keep what?Sui: keep fatigue away, that means you will get less stressful and more relaxing.Shan: maybe relaxing?Sui: Right! And …It can be seen from the above examples that L-L pairs used L1 in metacognitive and vocabulary strategies to quickly resolve communication problems during the interaction. However, with the aid of high-level peers, the H-L pairs produced more opportunities for negotiation of meaning through more interactional moves. This offered further evidence for the tendency that low-level students in L-L pairs produced more L1 words and turns, and fewer opportunities for negotiation of meaning, than those in the H-L pairs.The present study investigated the role of L2 proficiency pairing and task type in the amount and functions of L1 used by Chinese EFL learners engaging in dyadic task-based oral interaction. The dyadic interactional negotiation of the pairs was also examined.The participants in our study used L1 words in oral interaction to a fairly limited extent, at only 2.9%, lower than that in Storch and Aldosari . SimilarIn terms of L2 proficiency pairings, this study found a significant difference in the use of L1 words and turns between L-L pairs and all the other pairs in both tasks. This finding is inconsistent with those of Storch and Aldosari , 2012 anIn terms of task type effects, there was no significant difference in the amount of L1 words between different dyads. Our participants produced more minor L1 turns than predominant ones, particularly for opinion-exchange tasks. While H-H pairs produced more predominant L1 turns, the other pairs produced more minor L1 turns, with M-M and H-L pairs having produced far more minor L1 turns than predominant L1 turns. The analysis on L1 functions of H-H pairs showed that 50% of their L1 use, in predominant L1 turns, was off-task talk. These findings were similar to those of Azkarai and García Mayo, Our findings contribute to the existing literature by revealing the impacts of different L2 proficiency pairings on the amount of L1 use. A significant difference was found in the amount of both L1 words and turns between L-L pairs and all the other pairs  in both tasks. This finding indicates that low-level learners, when paired with other low-level learners, produced significantly more L1 words and turns than when paired with high-level learners. Early learner-learner oral interaction studies informed us that L-L pairs might produce more negotiated meaning (Pica et al., The second research question queried the functions of L1 use under different proficiency pairings and task types. We found that L1 served a wide range of functions to facilitate interaction and task completion, lending support to the findings of previous studies (Storch and Aldosari, Our findings could be explained from a sociocultural perspective (Vygotsky, In addition, Macaro et al.  proposedThis study also delved into the interactional negotiation of L1 functions by contrasting them with the functions served by L2, and demonstrated the effects of different proficiency pairings on the opportunities for negotiation of meaning through interactional moves. Although L-L dyads quickly resolved communication difficulties by employing L1 in metacognitive and linguistic strategies, with the aid of high-level peers, the H-L dyads produced more opportunities for negotiation of meaning than the L-L dyads. This finding lent support to previous findings that mixed-level dyads produced more interactional moves and opportunities for modified output than same-level dyads Porter, . Mixed-lWe need to acknowledge the limitations of our study. Firstly, we are unsure how much language learning or gains low-level learners achieved through peer interaction. Secondly, we only reported those selected interactional moves in those interactional episodes involving two L1 functions, metacognitive and vocabulary talk. Future research could examine whether and how low-level learners could make progress through peer interaction, and also quantitatively explore the causal relationship of L1 use and opportunities for negotiation of meaning.This study examined the effects of different language proficiency pairs and task type on the L1 use of Chinese EFL learners in dyadic task-based oral interaction. We found a significant difference between L-L pairs and other pairs in both the amount and functions of L1 use in both speaking tasks. Low-level learners produced significantly more L1 when paired with the same low-level peers than with high-level peers. Although the amount of L1 words and turns used by the L-L dyads was modest, fewer opportunities for negotiation of meaning were detected in their interactional episodes.The findings offer some pedagogical implications for EFL teachers about how to effectively implement dyadic task-based oral activities. Firstly, the two-way information-gap task and opinion-exchange role-play task were both suitable for EFL teachers to assign dyadic oral tasks without worrying about students generating too much L1 use. Secondly, EFL teachers could pair same-level learners of medium to high proficiency and mixed-level learners of high and low proficiency in L2 classrooms: this would not result in a significant increase in L1 use. It is better to pair low-level learners with high-level peers so as to produce more opportunities for negotiation of meaning and avoid generating significantly more L1 use, which might better facilitate learners' target language production and learning.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 School of Foreign Languages, Renmin University of China. The participants provided their written informed consent to participate in this study.LT conceived the initial idea, fine-tuned by YJ, designed the study, collected and analyzed the data, and drafted the manuscript. YJ collected and analyzed the data, revised, proofread, and finalized the manuscript for submission. All authors contributed to the manuscript and approved the submitted version.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.


This paper offers synchronic evidence supporting their grammaticalization. This evidence is based on two main conclusions: (1) these DMs share common grammatical items most of the grammaticalization sub-processes that lead to their emergence, and (2) they are not dis-integrated elements from the sentence grammar. They rather belong to the peripheral domain of grammar.The inclusion of discourse markers (DMs), which are commonly depicted as syntactically non-integrated elements, within the domain of grammaticalization has been a controversial topic in the last two decades. The emergence of DMs for some researchers are cases of grammaticalization, whereas they are viewed by some other researchers as elements that escape from the grammatical domain to the pragmatic and communicative domain. This paper argues with the grammaticalization of DMs. It provides evidence to this view from Jordanian Arabic. The cases under investigation in this paper are the DMs  Grammaticalization, Discourse marker, Discourse linker, Sentence grammar, Peripheral grammar, Jordanian Arabic One of the main challenges to grammaticalization is the implementation of its approaches on discourse material, including discourse markers (henceforth DMs).pragmaticalization is not necessary to capture the development of DMs due to the following observations: (1) many grammatical markers have pragmatic extensions and (2) many DMs submit to morpho-syntactic and phonological processes, which are generally typical processes in grammar. Another view presents DMs as the output of two independent stages; grammaticalization of content words followed by pragmaticalization  and the DM mahu is a product of a non-trivial coalescence of the DMs ma and hu. The discourse and pragmatic functions of these DMs in JA are also investigated. Concerning their discourse function, they are discourse linkers that mark a turn as responsive in the oral discourse. On the other hand, at the pragmatic/attitudinal level each of these DMs has its own peculiarity.From the previous discussion, it can be inferred that the development of DMs in a way analogous to that of common grammatical markers, such as auxiliary verbs, is controversial. This paper argues with the grammaticalization of DMs. It is to explore the emergence of three interrelated DMs in Jordanian Arabic (JA), namely, hu: ma:, their development into DMs and their discourse and pragmatic functions. It also explores the evolution and functions of the DM mahu. Section The outline of the current paper is as follows: Section 2In this section, the prototypical processes of grammaticalization are introduced and previous studies concerned with grammaticalization in Arabic are reviewed.2.1Grammaticalization is a multilayered process of language change that encompasses multiple semantic, morphosyntactic and phonological interacting processes that typically apply gradually . This reFollowing the desemanticization of a lexical word and the development of a new functional item, the functional item becomes prone to phonetic erosion. Phonetic erosion can be defined as the reduction of the phonological substance of the newly formed functional item in some way resulting in making it ‘more dependent on surrounding phonetic material’ . The com2.2ra:ħ ‘went’ and baddi ‘I want’ to future markers in JA. Similarly, ra:ħ as a future marker in Syrian Arabic. They report that this grammaticalization is paired with semantic, morphological, phonetic and morpho-phonological shift. Additionally, ʔabɣi ‘I want’ into a future marker. With regard to aspect markers, the development of some posture verbs to progressive aspect markers are studied in Kuwaiti Arabic . It may also appear in a post-verbal position where it is typically under focus, as exemplified in (1b). (PARTICIPLE = PART).The third person singular masculine pronoun at, does not agree with the masculine pronominal subject hu: in gender.As shown in (1a), the verb must agree with this pronoun in phi-features, akin to other pronouns. Any mismatch results in ungrammaticality. The cause of the ungrammaticality of the sentence in (2) is that the verb, which is marked with the feminine suffix -hu: developed a grammatical function: it acts as a pronominal copula in present tense sentences in JA and in some other Arabic vernaculars, such as Egyptian Arabic . Hence, the copular hu: is strongly contingent on the host structure; that is to say, it must reflect the phi-features of the subject in equational and predicational sentences and occupies a fixed position in these sentences. (SUPERLATIVE = SUPER).Additionally, n Arabic , 1991 ann Arabic  , the copula hu: agrees with the masculine singular subject sabab. Once the order of the singular topic and the plural complement is reversed, as shown in (4b), the copula is re-specified as plural to agree with ʔil-dʒamahi:r.What also argues with the development of the pronoun hu:, but some of them lack its copular counterpart, such as Modern Standard Arabic , hu: is located at the left edge of the sentence to create a yes/no question.7The copular hu: licensed its development into a DM, as DMs typically precede the sentences they scope over. The importance of syntactic position in licensing grammaticalization was previously reported in the relevant literature. hu: in initial position underwent phonological and morphosyntactic loss/reduction. It lost its stress, and then its long vowel underwent reduction. Further, it lost its ability to inflect: it cannot agree with the feminine subject ʔil-fikrah. Note that its presence is also optional, as marked by parentheses.This initial position of the interrogative hu in (6) is a DM that adds no semantic contribution to the proposition of the sentence over which it scopes.Given this optionality and its inability to have agreement in phi-features, the reduced 3.1.2hu acts as a discourse-linker in oral communications. It regulates the relation between turns. It marks an utterance it scopes over as non-initial  and ties it to the ongoing conversation. In (7b), the turn starts with the DM hu. It operates over the entire utterance and links it to the utterance in the previous turn in (7a). On the other hand, in the absence of the DM hu, the utterance in (7b) can be either an initiative or responsive turn in the conversation. The DM hu can also link a responsive turn to a prior action/event that happened in the universe of the oral discourse. For exemplification, while the interlocutors in (7) are watching a lot of people rushing down the streets, Speaker 2 can react to this event, which is known for both interlocutors, with the utterance in (7b) even if Speaker 1 does not initiate the conversation. This entails that the DM hu marks an utterance as a response/reaction to former linguistic material or a previous event in the discourse universe.At the discourse level, the DM hu adds more emphasis on the proposition of the declarative sentence it scopes over when the sentence offers a different opinion from that in the previous turn. This function is common in the presence of a modal-like item in the sentence, such as the adjective la:zim ‘necessary’. In (8), the interlocutors are discussing the reaction of Speaker 1 to an event. Speaker 2 starts his turn with the DM hu to emphasize that the decision that has been made by Speaker 1 is wrong and spontaneous.At the pragmatic/attitudinal level, the DM hu with imperatives or interrogatives has a persuasive function. In (9), it occurs before the imperative ʔuskut to show that the speaker is insisting on stopping shouting. In (10), the DM hu to the left of the question indicates that this question has been repeated by the speaker, and the function of the DM is to persuade the hearer to answer.Besides, the DM hu is proposed in (11). From the pronominal system in JA, the pronoun hu: was targeted to be developed into a copula and an interrogative operator, and then into a DM.Based on the previous discussion, the path that led to the emerging DM hu from the copula/interrogative operator and the development of the latter from a pronoun can be further supported by their synchronic properties. The common denominator among the pronominal, copular and discourse hu(:) is the linking function. The pronoun links its host sentence to a particular person or thing in the real world. Likewise, the copular hu: links two words or phrases  is ungrammatical as the verb ma: evolved into a DM in JA.The next part is mainly to argue that the negative particle 3.2ma: into several types depending on its functions. It can be a interrogative particle, relativizer, negative particle, gerund producing, indefinite adjectival, indefinite exclamative or preventative. It can also be superfluous.ma: as a negation particle, which is common in JA, as exemplified in (13).In MSA, Arab grammarians sorted hu:, ma: developed into a DM in JA. At the phonological level, ma: also underwent reduction when it was dropped in the domain of DMs, as shown in (14b). It lost its stress, and then its long vowel was reduced (shortened). At the discourse level, the DM ma is a discourse linker. It marks a turn as responsive. As can be observed in (14b), it links a responsive turn to a preceding one. To illustrate, Speaker 1 in (14a) is asking Speaker 2 to work with him; however, Speaker's 2 reply indicates that he may accept, but he needs to know what is the nature of that work first. It is worth noting here that the DM ma, similar to the DM hu, can link a responsive turn to a certain action or event in the universe of the oral discourse.Similar to ma: that developed into a DM is the negation particle. To illustrate, the presence of the DM ma and the negative particle in the same position in (14b&15), should imply that the negation particle is the raw material of the DM ma.In terms of distribution, the example in (14b) may indicate that the type of ma: and its DM counterpart do not have shared features. More specifically, the negation particle contributes to the propositional content of the sentence, whereas the DM is semantically null and has a communicative function. However, what supports the view that the negation particle is the source of the DM is that the meaningful content of the negation particle was not fully bleached once it was developed into a DM. The negation meaning has persisted in the DM counterpart at the pragmatic level: the DM ma, in (15b), is basically to reject a previous proposition and replace it with that of the utterance over which ma operates. To illustrate, Speaker 2 rejects to work with Speaker 1 immediately. He wants to know more about the nature of this work first. Hence, the DM ma, which is a kin to but in English, is basically to express (temporary) refusal at the level of the oral discourse. Another example illustrating this pragmatic and communicative function is the one in (16). Speaker 2 disagrees with Speaker 1 who has suggested leaving to another workplace in the night shift. Speaker 2 refuses leaving the workplace as he and all his friends agreed to finish the work, and then they may go to another workplace.At the first sight, the negation particle ma should be developed from the negation particle ma: and the negative connotation persists in the DM counterpart. The path of the DM ma is suggested in (17):Based on the previous discussion, the DM hu and ma is investigated.Below, the non-trivial coalescence of the DMs 3.3ma and hu were coalesced to form another DM, mahu. It should be highlighted first that this coalescence is not a case of two simple DMs superficially combined together only at the phonological level. It is rather a fully-fledged DM, as its components cannot be reversed or separated by an intervening element, as shown in the ungrammatical structures in (18).In JA, the DMs mahu links the utterance to the previous turn. At the pragmatic/attitudinal level, mahu is strongly persuasive, in comparison with the persuasive function of the DM hu. To clarify the persuading function of mahu and to compare it with that of the DM hu, consider the example in (19). In (20b), Speaker 2 wants to know the name of the person who told Speaker 1 that speaker 2 is traveling soon. Given the fact that it is the first time Speaker 2 requesting the name in (19b), he does not use any DMs. On the other hand, the use of the DM hu in (19d) indicates that Speaker 2 is repeating the same question after the ambiguous response of Speaker 1 in (19c). Speaker 2 is insisting one Speaker 1 this time to know the name of that person. Finally, Speaker 1 is trying his best to avoid mentioning that name in (19e), and the use of mahu in (19f) entails that Speaker 2 is over-insisting on his initial request and shows that he is too concerned. Hence, the DM hu has a persuading function, but mahu is strongly persuasive. As for the speaker's attitude, hu shows that he is concerned, whereas mahu denotes that he is too concerned and anxious.At the discourse level, hu, ma and mahu are discourse linkers with some variations in the attitudinal/pragmatic function(s). A grammaticalization path of these DMs is suggested in (20).To conclude this section, the DMs 4As introduced in Section hu:, which is specified as third person masculine singular, lost its phi-features once it was evolved into the DM hu, thus the DM hu does not refer to a particular person or thing and does not have agreement with any element in the sentence it scopes over. This type of semantic loss is common when typical grammatical(ized) items undergo further grammaticalization . They tend to lose the ability to inflect  where the DM ma that evolved from the negative particle ma:. In this example, the DM ma involves a negative connotation . Additionally, the linking function of the copular hu remains after its evolution to a DM and covers a communicative meaning. More specifically, the copular hu: and its DM counterpart have a linking function; however, the former links a complement to its subject and the latter links an utterance to the ongoing discourse. This implies that the functions of these DMs, albeit pragmatic, attitudinal and communicative, signify their development from other items.This section is to argue that the three target DMs in JA are cases of grammaticalization. What supports their grammaticalization is that they underwent some prototypical sub-processes of grammaticalization: desemanticization and phonetic erosion. inflect . Similarhu: and the negation particle ma: into DMs is accompanied by the loss of some of their phonetic material . Worth noting is that the disyllabic coalesced DM mahu can be reduced into monosyllabic mu in some JA sub-varieties.With regard to phonetic erosion (reduction or attrition), the evolution of the copular Another important point that supports the view that the development of the three DMs are cases of grammaticalization is paradigmaticity, which is a process previously associated with grammaticalization : 367. Wihu:, which should belong to core grammar, in a descriptive sentence in JA will not cause ungrammaticality of the host sentence, as can be observed in (21) below. Hence, obligatorification is not mandatory not only in the development of DMs, but also while forming some other grammatical items. This emphasizes on A point that may ostensibly argue against the grammaticalization of DMs is related to the stage of obligatorification (or obligatoriness), which is introduced by hu: is not rule-based (not governed by grammar); however, when it is present, grammar obligatorily sets its inflectional morphology . Similarly, the DM counterpart is not conditioned by inflectional grammar ; however, its syntactic position in the turn is governed by rules: ma and hu, for example, are not allowed to occupy a sentence/turn-final position, whereas the DM mahu is allowed to appear in this position.Obligatorification arises once the copula or a DM surfaces in a sentence/utterance nonetheless. It is true that the (non-)presence of the copular 5The aim of this section is to provide syntactic evidence to the integration of the three target DMs in sentence grammar. More specifically, they can interact with grammar-peripheral elements. In addition, the impact of this interaction on the information status of a host utterance is investigated.hu, it has been shown in Section hu can precede or follow peripheral materials, such as ‘style disjuncts’ following hu can either precede or follow the peripheral style disjunct badd-ak ʔil-sʕara:ħah in (22a) and the topic ʔil-matʕaʕi:m in (22b). The DM hu cannot appear between the integral elements of a sentence and in final position when these elements are kept in situ, as marked by an asterisk in (22). This implies that this DM is not an integral element in sentence grammar; however, it is visible to syntactic operations ).Starting with the DM ma should be integrated in sentence grammar due to its clitic nature. Specifically, it can combine with a referring pronoun from the target sentence, such as the first person singular pronominal clitic -ni in (23). Note that the pronoun combined with ma must enter in agreement with one of the elements in the sentence in phi-features. A subsequent process to this combination is that a constituent from the sentence grammar can move up in the syntactic structure above this DM, such as ʔil-ʃarikah in (24b). It does not go without mentioning that the entire sentence can appear to the left of ma-hi, as in (24c). This indicates that it has been moved up in the syntactic representation above the DM ma-hi. Note that the combination of the DM ma with a referring pronoun (a part of the sentence grammar) licenses its presence in any position in a sentence even in the absence of any movement in the syntactic structure, as marked between parentheses in (24).Similarly, the DM mahu, comparable to the previous DMs, it can appear to the right or left edge of peripheral elements, such as style disjuncts or moved constituents. Hence, it can appear in various positions in an utterance: in utterance-initial position in (25a), to the right of a moved constituent in (25b) and in utterance-final position in (25c) after moving the entire sentence above this DM in the syntactic structure. However, it cannot be located between the integral elements of the sentence, which are in situ, as marked in (25d).With regard to the DM ma has a clitic nature, and consequently it may appear in various position in sentence grammar.To wrap up, it has been shown that the three DMs can be accounted for in sentence grammar. They are visible to syntactic movement, and therefore they should belong to the domain of peripheral grammar. Note that the DM mahu, its presence early indicates that the information content of the host utterance is new, whereas in final position it is given. Consider the example in (26) where mahu in Speaker's 2 turn-initial position is used to presuppose that Speaker 1 does not know this information. On the other hand, its presence in turn-final position signifies that Speaker 2 presupposes that Speaker 1 knew this information about his car, and therefore it can be understood from this turn that Speaker 2 is blaming Speaker 1 for his carelessness.In addition to the interaction of these DMs with peripheral elements in sentence grammar, the position of a DM can have an impact on the information content of the host utterance. Starting with the DM ma combined with a referring pronoun. In the presence of mahinne in turn-initial position in (27b), the information content is new, whereas in turn-final position, it is old.Similar conclusions can be said about the DM hu, it does not appear in turn-final position. In this position, short forms are lengthened. From a prosodic perspective, this position is typically marked with utterance-final lengthening . The presence of the DM hu in this position will result in recovering the full form of its source material  by vowel lengthening, as shown in (28). Hence, the ambiguity whether this item is a pronoun/copula or a DM in utterance-final position, prevents the occurrence of the DM hu in this position.With regard to the DM ma, it has a clitic nature and should not be stranded alone in utterance-final position. Further, its lengthening into ma: in this position causes the same confusion/ambiguity of leaving the DM hu in final position.As for the DM Depending on the previous discussion, although the three DMs in JA are optional and have metatextual and pragmatic functions, they should not be considered syntactically non-integrated elements as their development is characterized with the most common processes of grammaticalization and they can be accounted for within sentence grammar . Hence, the three DMs in JA should be subsumed within the realm of grammar and grammaticalization.6hu, ma and mahu for the following reasons. First, their development is indiscernible from the proper grammaticalization processes. It involves desemanticization, phonetic erosion and paradigmatization. Second, they are not disintegrated elements from sentence grammar. More precisely, they should be considered paraphrial elements within sentence grammar, as they are visible to syntactic operations . Thus, they belong to the domain of peripheral grammar. Third, they influence the information structure of a host sentence. Hence, data from JA cast doubts on the proposal that the grammaticalization of DMs can be visualized as an escape from the domain of grammar, either core or peripheral. They rather argue with maintaining the development of DMs within the realm of grammaticalization.The current paper has argued with the view that the development of DMs should be studied as cases of grammaticalization, as previously proposed in . In thisAbdulazeez Ahmad Jaradat: Conceived and designed the analysis; Analyzed and interpreted the data; Contributed analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


Friction and wear of polymers at the nanoscale is a challenging problem due to the complex viscoelastic properties and structure. Using molecular dynamics simulations, we investigate how a graphene sheet on top of the semicrystalline polymer polyvinyl alcohol affects the friction and wear. Our setup is meant to resemble an AFM experiment with a silicon tip. We have used two different graphene sheets, namely an unstrained, flat sheet, and one that has been crumpled before being deposited on the polymer. The graphene protects the top layer of the polymer from wear and reduces the friction. The unstrained flat graphene is stiffer, and we find that it constrains the polymer chains and reduces the indentation depth. Graphene is a two dimensional material that has remarkable properties, both electronic –2 and meThe tribological properties of polymers coated with graphene have barely been studied on the nanoscale due to the added complexity of the polymer, the tribology of which, even without any coatings, is still not well understood –23. In eIn this work, we aim to develop our understanding of the frictional behavior of a polymer coated with graphene by using molecular dynamics simulations of a single sliding asperity at the nanoscale. We show that graphene protects the polymer substrate from wear and identify the mechanism of this protection. We show that crumpling of the graphene has an impact on the friction. In the next section we first describe the simulation setup. Then we move on to discussing our simulations of depositing, indenting, and sliding on graphene. In the final section, we draw some conclusions.We simulate a slab of polyvinyl alcohol (PVA) coated with a single layer of graphene and a counterbody representing an AFM tip consisting of silicon. The simulations were performed using LAMMPS . We use 2H4O) (see PVA is described using a united-atom force field developed by Müller-Plathe and co-workers . Each poH4O) see . The non0 = 0.0179 eV, σ0 = 4.628 Å, and r is the distance between the interacting monomers. The bonded interactions are described by a harmonic potential Vbond = K(r − r0)2, where K = 2.37 eV/Å2 is the stiffness and r0 = 2.6 Å is the equilibrium bond length. The bending potential is approximated by an angular potential described in a table format.where εFor graphene we use the AIREBO-M potential developed by O’Connor and co-workers . It is a1 = 4.025 Å, and ε1 = 0.015066 eV. We model the interaction between the silicon tip and graphene using a Lennard-Jones 12-6 with the same parameters used by Li and co-workers [2 = 3 Å is the distance at which the potential is equal to zero. In our system, the tip and polymer are never in direct contact. They are always separated by graphene. We therefore do not need to model their interactions. However, to be sure that no extremely unphysical events can occur, we have used the same potential as for the non-bonded polymer–polymer interaction.The interaction between PVA and graphene is modelled using a Lennard-Jones 12-6 potential and the Lorentz–Berlot mixing rule, so that σ-workers . ε = 0.0The masses of the particles were chosen to be equal to 12.01 g/mol for the carbon atoms of graphene, 44.17 g/mol for the monomers in PVA and, 2.8 g/mol for the particles of the AFM tip. This leads to a fairly small total tip mass. While this is not entirely physical, such a low mass will help speed up the dynamics and damping of the tip and save computation time without compromising the results . We simux- and y-directions , filled up with PVA molecules placed randomly and constrained by hard walls in the z-direction. The box contains about 250,000 monomers in chains of 50. Because there are overlaps, we initially give them no interaction. To remove overlapping gently, we first applied a nonphysical soft hybrid interaction potential, for 0.25 ns to remove particle overlapping, and then slowly ramp up the potential over a period of 0.25 ns to the coarse-grained potential described in the previous section. The hybrid interaction potential consists of a 12-6 Lennard-Jones potential for the non-bonded interactions and a spring potential for the bonded interactions.We start from a box with periodic boundary conditions in the z-direction as they are no longer needed. The density at this point is 22 monomers/nm3. We also switch the thermostat to a Langevin thermostat with a temperature of 300 K and a damping time of 0.1 ps. This thermostat is applied only to the bottom quarter of the PVA molecules, and later to the graphene sheet.Once we have reached a melt with the correct interaction, we equilibrate it for 0.25 ns in the NVE ensemble. The temperature of the melt at this point is extremely high. To obtain a realistic semicrystalline substrate structure, we cool down the sample using a Nosé–Hoover thermostat with a linearly decreasing temperature, starting at 5000 K down to 220 K with a cooling rate of 75 K/ns. The damping time of the thermostat is 0.1 ps. After this, the temperature is kept constant at 220 K for 4 ns. At this point, we remove the walls and the 2.To prevent the polymer slab from moving as a result of the external forces during deposition of the graphene, indentation, and sliding, the centers of mass of the chains in the lower quarter of the substrate are tethered to their original positions using springs with spring constant 1 eV/Åx- and y-directions by 10%, which leads to wrinkles on the surface  for a period of 75 ps, after which it sits on the surface and has stopped moving. The total normal force applied is around 4 nN (3.3 MPa). Then the force is removed and the graphene sheet stays on the surface due to adhesion.In both cases, we deposited graphene on the surface of the polymer substrate by placing the graphene sheet at around 90 Å from the surface and then applying a force to each of the graphene carbon atoms equal to 0.00005 eV/Å , we start the sliding. To better highlight the influence of the tip radius, we average the frictional forces between support displacements of 50 and 100 Å. We plot those results as a function of the normal load for two different tip sizes (radius of 50 and 100 Å) in In the case of the crumpled graphene sheet , the friWe compare this to sliding without graphene. In a simulation with no graphene, a normal load of 51 nN, and a tip radius of 100 Å, we found that the tip moves deeply inside the substrate and the average friction force is above 90 nN, almost an order of magnitude higher than with graphene. This clearly shows that the addition of the graphene layer drastically reduces the friction.To further investigate the mechanisms and observe the effect of sliding on the wear of the polymer material, we compare three simulations, that is, one without graphene, one with flat graphene, and one with crumpled graphene. In this way, we can distinguish between the short-range effects of the protective outer coating, and the more long-range mechanical effects of the graphene layer on the shape of the surface. A normal load of 1 nN and a tip radius of 50 Å were used in all simulations .To improve the averaging by increasing the total sliding distance, we increase the sliding speed by a factor of 10 to 20 m/s. The displacement vectors are recorded after 0.6 ns, meaning that the support has moved 120 Å. This is indicated by the dashed and solid lines. Without graphene, the vector displacements close to the surface are high and in the sliding direction. This indicates that strong residual deformation remains at the surface because of the shearing of the chains. We observe that the displacements of the polymer are roughly an order of magnitude less when graphene is present. This indicates that graphene efficiently prevents damage of the substrate. The displacements are the smallest in the case of the flat graphene sample, where the graphene sheet is not just protecting the polymer from the tip, but also constraining the chains. Our work thus shows that it is the interaction between graphene and polymer that plays a crucial role in the friction and wear.The graphene layers we have used, both flat and crumpled, are constrained to remain at a specific length because of the periodic boundary conditions of the simulation box. This means that any elastic stretching of the graphene sheet is limited to a fairly small area. In reality, most of the graphene sheets are larger than the length of our simulation box and depending on how they attach to the surface, they may thus have more length to stretch elastically. Our crumpled graphene sheet, by having a longer equilibrium length than the box, is more representative of completely unconstrained, loose graphene sheets. However, graphene that is bound to the polymer surface, through adhesion or covalent chemical bonds, would behave more like the flat graphene in our simulations and would provide additional protection.The temperature dependence of friction can potentially allow one to probe different relaxation mechanisms in the polymer –34. In okBT/ps of power. Given the density and the fact that the typical time scale of dissipation is around 1 ps, this is sufficient to increase the temperature of the substrate locally by several kelvin. Near the glass transition, this could lead to significant changes in yield strength. The sinking affects the real surface area and thus also has a noticeable effect on the friction when the normal load is high. The graphene sheets reduce the wear by both reducing indentation and constraining the chains. The displacements of the chains are roughly an order of magnitude smaller when a graphene sheet is present compared to the case with no graphene. The graphene is curved away from the tip; this is especially true for the flat graphene layer. This helps to spread out the pressure and to reduce the local pressure in the polymer. The flat graphene sheet is the most efficient at reducing the friction and wear of the system by this mechanism, as it is harder to penetrate.We investigate the effect on friction and wear of a graphene coating on a polymer by simulating friction force microscopy experiments with molecular dynamics. A rigid counter-body simulating the tip of the AFM is rubbed against a substrate made of a semicrystalline polymer (PVA) with a graphene sheet on top. Two different graphene sheets have been investigated, that is, a flat graphene sheet that has the same size as the simulation box and a crumpled graphene sheet that has been biaxially compressed by 10%. This allows us to probe some of the mechanisms at play in these systems. Before and after the sheet is deposited on the substrate, we computed the roughness. We observe that the crumpled graphene sheet accommodates to the roughness of the polymer, while the flat graphene sheet reduces the roughness. We also observe a rearrangement of the chain near the surface into a layered structure, indicating that the chains tend to align parallel to the surface. During sliding, the tip sinks slowly into the material. We speculate that this is related to frictional heating in the substrate. For the tip of 100 Å, the sliding delivers about 6


Recent advances in nanoscience have opened ways of recycling substrates for nanomaterial growth. Novel materials, such as atomically thin materials, are highly desirable for the recycling substrates. In this work, we report recycling of monolayer graphene as a growth template for synthesis of single crystalline ZnO nanowires. Selective nucleation of ZnO nanowires on graphene was elucidated by scanning electron microscopy and density functional theory calculation. Growth and subsequent separation of ZnO nanowires was repeated up to seven times on the same monolayer graphene film. Raman analyses were also performed to investigate the quality of graphene structure along the recycling processes. The chemical robustness of graphene enables the repetitive ZnO nanowire growth without noticeable degradation of the graphene quality. This work presents a route for graphene as a multifunctional growth template for diverse nanomaterials such as nanocrystals, aligned nanowires, other two-dimensional materials, and semiconductor thin films. Epitaxy of thin films and nanomaterials is a crucial step for device manufacturing. However, conventional epitaxy is preferably conducted when original substrates and overgrown materials possess similar lattice constants and thermal expansion coefficients. To utilize the epilayer, the substrates are mostly disposed of or used as a base substrate in devices. Since the cost of substrates for epitaxy process takes up a significant portion in device manufacturing, there has been an intensive research effort to recycle substrates. Epitaxial lift-off (ELO) is a technique of recycling wafers , a typicGraphene provides novel opportunities for substrate recycling because of its self-terminated surface without surface dangling bonds, which usually act as origins of interfacial defects and strong chemical bonding between graphene and an overgrown material. Successful demonstrations of van der Waals epitaxy and remote epitaxy show that graphene is a versatile substrate for epitaxial growth of elemental and compound semiconductors ,9,10,11.In this work, we report a recycling process of monolayer graphene used as a growth template for synthesis of single crystalline ZnO nanowires (NWs). The single crystalline ZnO NWs were selectively grown on the graphene. Moreover, the selective nucleation of ZnO on graphene was investigated by first-principles calculations based on density functional theory (DFT). Hydrothermal synthesis and delamination processes using chemical exfoliation of the ZnO wires were repeated on a same graphene layer. Raman spectroscopy analysis demonstrates that the graphene layer remains stable after multiple NW synthesis and delamination processes. In addition, Raman mapping analysis was performed to investigate the quality of graphene surfaces along repetition of ZnO NW growth and subsequent delamination. The chemical robustness of graphene enables the reuse of the graphene layer for repeated nanomaterials growth (at least seven times) without noticeable degradation of its quality. This work provides a route for graphene as a recyclable growth template for various materials.2 atmosphere. Next, methane  are introduced in the system while the temperature was increased to 1020 °C for 20 min. Lastly, the sample was rapidly cooled down to room temperature. The synthesized graphene sheet with a surface area of 1.5 cm × 1.5 cm was transferred onto a SiO2/Si substrate using a PMMA-free transfer method [Monolayer graphene was synthesized on a 0.1 mm thick Cu foil  using a low-pressure chemical vapor deposition which was previously reported . The Cu r method . The tra® 495 K C3, Newton, MA, USA) layer was deposited by spin coating. The EBL was performed using the JEOL 6300 FS instrument . The e-beam exposed PMMA regions defined as dot patterns were removed by development in methyl isobutyl ketone (MIBK):isopropyl alcohol (IPA) solution. The remaining PMMA layer acted as mask of selective growth of ZnO NWs on the exposed graphene.E-beam lithography (EBL) was employed to define the nucleation sites of ZnO NWs on the graphene sheet. A 300 nm-thick poly (methyl methacrylate) 2·6H2O), hexamethylenetetramine , and polyethylenimine  in deionized water. Zinc nitrate hexahydrate and HMTA were employed as precursors to form ZnO. PEI was used to induce growth along one principal direction by adsorbing onto nonpolar sidewalls of ZnO crystal. The synthesis was performed at 95 °C for 4 h. Details of the hydrothermal growth procedure are reported elsewhere [ZnO NWs were grown using hydrothermal method on the graphene/SiOlsewhere .Raman analyses for point spectra and mapping were taken by a homebuilt system, which was calibrated using a Si wafer. A frequency doubled 532 nm Nd:YAG laser was used to probe the nanostructures and the Raman signal was collected via a liquid nitrogen cooled CCD detector . For the micro-photoluminescence (PL), a frequency quadrupled Nd:YAG laser with the wavelength of 266 nm, a pulse width of 400 ps, and a repetition rate of 10 kHz was employed as the excitation source.2 surface. Position-controlled hydrothermal growth of ZnO micro/nanostructures on graphene has been reported by several research groups [2 or graphene were formed to reduce required computational resource and approximate the binding energies between ZnO and the substrates without consideration of edge-effects. The total energies of supercells of ZnO, SiO2, and graphene were calculated based on DFT as implemented in the Vienna Ab-initio Simulation Package (VASP). All calculations were conducted with projector augmented-wave (PAW) potentials using the generalized gradient approximation (GGA) of Perdew, Burke, and Ernzerhof (PBE). Each model system was relaxed along the z-direction using preconditioned residual-minimization until the force on the atoms was less than 0.01 eV/Å. In these calculations, no edge or vacuum effects were considered.The surface morphology and the microstructures of the ZnO NWs were investigated by scanning electron microscopy  and transmission electron microscopy . h groups ,22,23. Eh groups . Our obsEsubstrate is the total energy of the substrate, SiO2 or Graphene, EZnO is the total energy of the ZnO nanorod, EZnO/substrate is the total energy of the combined nanorod/substrate system, and n is number of Zn or O atoms at the surface of the substrate. The binding energies were calculated to be 0.3654 and 1.7864 eV for the ZnO/SiO2 and ZnO/graphene structures, respectively. The higher binding energy for ZnO/graphene compared to ZnO/SiO2 indicates that the growth of ZnO nanostructures is energetically more favorable on graphene than on SiO2 [The relative binding energy per Zn atom is defined as on SiO2 .c-oriented ZnO NW is single crystalline. The formation mechanism of sharp tips of the ZnO NWs can be attributed to surface energy anisotropy that (0001) c-plane has the highest surface energy in wurtzite ZnO. Hence, reduction of the surface area of (0001) plane is energetically favorable [TEM was used to investigate the structural characteristics of the ZnO NWs on graphene. avorable .2 region was negligible. High crystallinity and dominant free excitonic emission in RT µ–PL spectrum of the ZnO NWs demonstrate that graphene can be employed as a growth template for high-quality ZnO nanostructures.The optical properties of the ZnO NWs were investigated using micro-photoluminescence (µ–PL) spectroscopy at room temperature (RT). The strong PL emission centered at 3.25 eV on the graphene, shown in 2, and Si, the repetitive growth and etch process can be successfully utilized to recycle the growth templates.To recycle graphene layers and repeatedly synthesize ZnO NWs, repetitive detachment processes of ZnO NWs are required. Mechanical exfoliation is an alternative way to separate the semiconducting materials from graphene surface. However, the mechanical exfoliation method easily damages either ZnO or graphene layer. Hence, we developed a wet chemical delamination process to simultaneously preserve the original structure of the ZnO NWs and that of the graphene layer. The slanted ZnO NWs b result e-beam lithography. The graphene area was patterned to check the quality of the graphene layer on a defined spot during repetition of the cycle consisting of growth and separation of ZnO NWs. 2/Si substrate. The observation of selective growth of ZnO NWs on the exposed graphene areas is consistent with the result shown in −1, which can be assigned to an A1g breathing mode originating from disorders, to the high frequency E2g, and to the second order of the D, respectively [Raman spectroscopy is a non-destructive technique commonly used to probe various carbon nanostructures ,30,31. Tectively . Raman aectively ,33. The ectively . To mainectively ,34.The Raman spectra shown in Raman mapping clearly reveals that ZnO NWs were grown on graphene as shown in −1 and no change in the relative intensities of the three major bands  were observed. The slight shift in values of ±15 cm−1 is likely due to environmental or equipment differences on the collection days instead of changes to the graphene structure [A key feature of recycling graphene for semiconducting nanomaterial growth is the remarkable properties that this 2D material continues to possess without degradation when repetitively used as a substrate. Thorough Raman spectroscopy analyses including point spectra and Raman mapping were performed over the growth area to characterize the quality of graphene along the repetition of ZnO NWs growth and subsequent delamination. The process of growth and delamination of ZnO NWs was repeated additional seven times on the same graphene. The overlays of all eight spectra of the graphene after each recycling process are shown in tructure . Raman structure ,39.2 was conducted. Raman maps of G, and 2D bands in To further characterize the graphene structure after each growth and removal of nanowires, Raman mapping in a graphene area of 150 µm2 layer, as shown in the inset of Structural and optical properties of the ZnO NWs grown at the different cycles of the growth-etching process were also characterized. No noticeable degradation of materials quality in both recycled graphene and ZnO wires region shows that the recycling graphene process in this study is applicable to production of nanomaterials without concern of process-dependent material degradation.Recycling of graphene as a growth template for single crystalline ZnO NWs was demonstrated by hydrothermal synthesis and chemical wet etching processes. The crystallinity and position-controlled growth of ZnO NWs were investigated by the SEM, and TEM analyses and interpreted by theoretical calculations. Raman analyses demonstrate that the graphene template was very stable and reliable after the repetitive growth and detachment processes. Our developed strategy for template-assisted synthesis of nanomaterials based on CVD graphene and recycling of the templates opens up a path forward for this graphitic material as an exciting growth substrate not only the research field, but also manufacturing and industrial fields.


NAC  transcription factors (TFs) play vital roles in plant development and abiotic stress tolerance. Salt stress is one of the most limiting factors for rice growth and production. However, the mechanism underlying salt tolerance in rice is still poorly understood.OsNAC3 expression in roots. Immunostaining showed that OsNAC3 was localized in all root cells. OsNAC3 knockout decreased rice plants’ sensitivity to ABA but increased salt stress sensitivity, while OsNAC3 overexpression showed an opposite effect. Loss of OsNAC3 also induced Na+ accumulation in the shoots. Furthermore, qRT-PCR and transcriptomic analysis were performed to identify the key OsNAC3 regulated genes related to ABA response and salt tolerance, such as OsHKT1;4, OsHKT1;5, OsLEA3–1, OsPM-1, OsPP2C68, and OsRAB-21.In this study, we functionally characterized a rice NAC TF OsNAC3 for its involvement in ABA response and salt tolerance. ABA and NaCl treatment induced This study shows that rice OsNAC3 is an important regulatory factor in ABA signal response and salt tolerance.The online version contains supplementary material available at 10.1186/s12870-021-03333-7. Oryza sativa L.) is the major staple cereal food for over one-third of the world’s population. Unfortunately, rice yield and grain quality are often affected by adverse environmental factors such as salinity, drought, or inappropriate temperature [Rice  family of TFs has acquired great attention for their role in abiotic stress resistance . So far,entified . Normallentified . NAM fro[petunia  and CUC2bidopsis  are one bidopsis . For exasduction . OsNAC2 sduction . Downrege grisea . ABA, sanic rice . The RD2bidopsis .OsNAC2 confer to drought and salt resistance and ABA biosynthesis gene OsNCED3 is upregulated [OsNAP [Apart from stress-related TFs that directly regulate the expression of abiotic stresses related genes, phytohormones such as ABA also play a critical role in plant abiotic resistance . Stress egulated . OsNAP ed [OsNAP . Transged [OsNAP .OsNAC3 and physiological phenotypes using its knockout and overexpressing transgenic rice lines. High throughput RNA-seq assay was performed to find the downstream target genes of OsNAC3. We show that OsNAC3 positively regulates the ABA pathway and salt tolerance in rice via the regulation of OsHKT1;4, OsHKT1;5, OsLEA3–1, OsPM-1, OsPP2C68, and OsRAB-21. These findings can help the development of better salt-tolerant crops.Previously, OsNAC3 was suggested to be involved in rice stress responses . HoweverOsNAC3 (Os07g0225300) was cloned based on the Rice Annotation Project Database . We used the OsNAC3 amino acid sequence as a query to search for its homologs in rice and Arabidopsis, and 17 homologs with more than 75% identity were obtained. These proteins, together with OsNAC3 and OsNAC45, were used to construct the phylogenetic tree. Phylogenetic analysis showed that OsNAC3 is closely related to the NAC family of TFs (SNAC1 and OsNAC4) in rice sites, and three As-1 sites  treatment in a time and dose gradient manner. Notably, OsNAC3 expression was the highest at 12 h and showed a positive correlation with the concentration of NaCl  was observed in the whole cell, while the green signal of OsNAC3 nicely overlapped with the red fluorescence of OsGhd7-RFP  and introduced it into rice protoplasts along with nuclear marker RFP Fig. A-H, indiOsNAC3-OsNAC3-GFP transgenic rice was constructed. Immunostaining assay was performed with GFP and DAPI was used as a nuclear stain. Antibodies against GFP  were detected in all the cell layers of transgenic roots, while it nicely overlapped with DAPI stain  in the nucleus  only grew on the SD/Trp-, but not SD/Trp−/His−/Ade- plates  with CRISPR/Cas9 method and a maize ubiquitin promoter-driven OsNAC3-overexpression line   for two weeks. We found that in the absence of NaCl, the growth of WT and OsNAC3- transgenic plants did not show a significant difference. However, in the presence of 75 mM NaCl, MT lines showed more withered leaves and less dry weight compared with WT lines; on the contrary, OE lines displayed attenuated chlorosis symptoms and greater dry weight  and salt (75 mM NaCl) conditions. Under control conditions, tissue Na+ and K+ content showed no differences between the WT and osnac3 lines  that of WT plants, while no significant difference was found in the roots. Besides, K+ concentration in roots of two MT lines was only slightly higher than that of the WT plants, while showed no significant difference in the shoots  rice roots under normal and salt conditions. A two-fold change in expression was selected as the threshold to determine the DEGs between the WT and MT lines under normal and salt conditions. We found that there were 3184 up-regulated and 1401 down-regulated DEGs between the WT and MT lines under normal and salt conditions, respectively  and mutant OsRAB21(Os11g0454300), OsPP2C68 (Os09g0325700), OsLEA3–1(Os05g0542500), OsPM1 (Os05g0381400), OsSUB12 (Os02g0198700), OsHKT1;4 (Os04g0607600) and OsHKT1;5 (Os01g0307500), was lower in the MT than in WT plants  encoding an ABA influx carrier, which is reported to mediate ABA influx functioning in the ABA signaling pathway [OsNAC3 and OsNAC45 transgenic rice under ABA treatment.To further investigate the role of OsNAC3 in rice ABA response, phenotypes of MT and OE seedlings were analyzed after exogenous ABA treatment. We found that the shoot and root length of OE plants was significantly suppressed compared with WT; on the contrary, the shoot length of MT plants was significantly more than that of WT plants. These results demonstrated that OsNAC3 plays important role in ABA signal response. In a previous study, we showed that another rice NAC transcription factor OsNAC45 was involved in ABA response. Under exogenous ABA treatment, T plants . Similar pathway . This soOsNAC3 knockout changed rice sensitivity to salt stress. Under 75 mM NaCl treatment, MT plants exhibited more wilting leaves compared to WT plants. Moreover, in the absence of NaCl, the roots and shoots concentrations of Na+ and K+ were similar between the WT and MT plants. In the presence of 75 mM NaCl, shoot Na+ concentration significantly increased in MT than in WT, while root K+ concentration was only slightly higher in MT. However, under the same treatments, tissue Na+ and K+ concentrations were similar between WT and osnac45 lines although OsNAC45 knockout lines also showed more sensitivity to salt stress than the WT plants. In osnac45 mutants, more accumulation of reactive oxygen species (ROS) in roots was detected [OsNAC3 and OsNAC45 might be involved in rice salt tolerance through distinct regulatory mechanisms. The RNA-seq and qRT-PCR analysis showed that the expression of some genes encoding Na+/K+ transporters such as OsHKT1;4 and OsHKT1;5 was regulated by OsNAC3. It is well known that HKT (High-affinity K+ Transporter) gene family mediate Na+ and K+ transport in rice [+ unloading in xylem to prevent over accumulation of Na+ in shoot [+ accumulation in MT shoots under salt conditions can be attributed to the lower expression of OsHKT1;4 and OsHKT1;5. These results indicated that OsNAC3 mutation changes the expression of some Na+/K+ transporters in roots causing less Na exclusion in roots and more Na root-to-shoot translocation, which in turn increases rice sensitivity to salt stress.detected . These d in rice . Under sin shoot –38. TherOsNAC3 knockout under normal and salt conditions. Go enrichment analysis showed that most DEGs were related to heme binding, tetrapyrrole binding, and oxidoreductase. To validate some of the DEGs and their homologs in rice, we performed qRT-PCR and found that OsLEA3–1, OsRAB-21, OsPM-1, OsPP2C68, OsHKT1;4, OsHKT1;5, OsSUB12 might be regulated by OsNAC3. OsLEA3–1 is an abiotic stress-induced gene, which encodes for a late embryogenesis abundant (LEA) protein. Overexpression of OsLEA3–1 significantly increases the rice tolerance to salt and drought stress [OsRAB-21 is upregulated to counter the salinity, drought, and ABA stresses [OsPM1 expression is regulated by OsbZIP46, which then plays important role in response to drought stress [OsPM1 expression is regulated by OsNAC45, indicating the regulatory role of OsNAC3 in the ABA signal transduction pathway along with OsbZIP46 and OsNAC45 [+ and K+ transport are proposed to be major players in root-to-shoot Na+ partitioning and K+/Na+ homeostasis [OsNAC3 involved in ABA response and salt tolerance might be due to regulate the expression of above-mentioned genes.Furthermore, transcriptome sequencing analysis revealed 1401 down-regulated DEGs in the roots after t stress , 40. Ricstresses , 42. OsPt stress . Recentl OsNAC45 . OsPP2C6 OsNAC45 . OsHKT1;eostasis –38. ThesOsNAC3. These results suggest that OsNAC3 might play important roles in multiple abiotic stress responses in rice.Interestingly, OsSUB12 (Submergence Tolerance 12), encoding an ethylene-responsive transcription factor, is homologous to OsSUB1A that mediates rice submergence stress tolerance . Further+ homeostasis in rice.In summary, OsNAC3, a novel nuclear-localized transcription factor, participates in ABA response and salt tolerance through regulating the expression of stress-responsive genes and shoot NaOryza sativa cv Nipponbare), two OsNAC3-knockout lines, and two OsNAC3 overexpression lines in this work. The wild type rice was obtained from rice resources conservation center of Guangxi University. The OsNAC3 mutants and overexpression lines were constructed in our laboratory (see below). The cultivation of plants conforms to China’s legislation on genetically modified plants. The formal identification of the OsNAC3-knockout and overexpression lines was conducted by Xiang Zhang and Yan long. Seeds of the wild-type, OsNAC3-knockout and overexpression lines were preserved in our lab, but not in a publicly available herbarium.We used the wild-type rice  as described previously [Rice seeds were germinated in water at 28 °C for 2 days in darkness. The germinated seeds were then placed on a floating net containing 0.5 mM CaCleviously .OsNAC3. 5-day-old Nipponbare was used to study the induction of OsNAC3. For salt stress, rice seedlings were exposed to different concentrations of NaCl  for 12 h or 100 mM NaCl for different times . For ABA treatment, rice seedlings were exposed to different concentrations of ABA  for 12 h or 50 μM ABA at different times .Root, stem, leave, leave sheath, and spike of heading stage rice were harvested for tissue-specific expression analysis of OsNAC3-OE construct, total RNA, extracted from Nipponbare, was reverse transcribed to cDNA by RT-PCR, which was then used as a template to amplify the OsNAC3 coding region. The full-length cDNA of OsNAC3 was inserted into the pCAMBIA1300-Ubi vector between the Ubiquitin promoter and nopaline synthase terminator. To create the OsNAC3-MT construct, CRISPR/Cas9 gene-editing system was used. The pCRISPR-OsNAC3 construct with two OsNAC3-specific target sites was constructed as reported previously [Agrobacterium tumefaciens strain EHA101, which was then transformed into rice cv. Nipponbare. The primers used in this work are listed in Supplementary Table 1.To create the eviously . These chttp://www.clustal.org/omega/) and graphical representation was created with Espript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/).Amino acid sequences of several stress-response NAC genes were compared with OsNAC3 using MEGA7 with 1000 bootstrap replicates and the neighbor-joining tree (NJT) method. Alignment of OsNAC3, ANAC102, SNAC1, and OsNAC4 proteins was performed with CLUSTAL OMEGA (https://www.ncbi.nlm.nih.gov/) and putative cis-elements were searched with Plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).For promoter analysis, 2000 bp upstream of the transcription start site were retrieved from NCBI , pGBK -OsNAC3, or pGBK-T7 empty vector (negative control); these vectors were then transformed into yeast strain AH109 according to the Matchmaker Gold Yeast Two-Hybrid System user manual (Clontech). Yeast cells carrying the pGBK- OsNAC3, pGBK -OsNAC45 or empty vector were spotted on SD/Trp- or SD/Trp−/His−/Ade- medium. The plates were photographed after incubation for 3 days at 30 °C.For transactivation activity analysis, the coding region of 2). Approximately 1 × 106 cells were used for the following transformation.The protoplasts were isolated based on the procedures described by Zhang et al.  with somOsNAC3 was fused in-frame to GFP of vector pYL322-GFP, producing the pYL322-OsNAC3-GFP construct. pYL322-OsNAC3-GFP, or vector control; these were co-transformed with nucleus marker (RFP-OsGhd7) into rice protoplast as described previously [To study the subcellular localization of OsNAC3, the coding region of eviously . After 1OsNAC3 without stop codon was PCR amplified from Nipponbare cDNA. Promoter and coding region were cloned into the pCAMBIA1300-GFP vector to generate the ProOsNAC3-OsNAC3-GFP construct, which was transfected into Agrobacterium tumefaciens strain EHA101 for transformation into rice cv. Nipponbare.A 2000 bp region upstream of the transcription start site was PCR amplified from Nipponbare genomic DNA; the coding region of OsNAC3-OsNAC3-GFP were embedded in 5% agarose. Next, cross-sections were generated with a micro slicer , and the sections were incubated with the rabbit anti-GFP polyclonal antibodies, followed by secondary antibodies  at room temperature (RT). Fluorescence images were captured by confocal laser scanning microscope ; DAPI was used to stain nuclei.Immunofluorescence assays were performed to detect cellular localization of OsNAC3 as described previously . BrieflyTo examine the growth rate of transgenic rice under ABA treatment, Nipponbare and transgenic rice seeds were germinated on 1/2 MS medium at 28 °C for 2 days in darkness. Then, the seedlings of similar size were transferred to 1/2 MS medium containing 0 or 2 μM ABA in a growth chamber at 28 °C and a 12/12 h light/dark cycle. Shoot and root length were measured after 5 days of incubation.3 at 130 °C [+ and K+ contents in the digested solution were measured by ICP-MS .To investigate the salt tolerance of transgenic rice, 4-leaf-stage transgenic and Nipponbare rice (14-days old) were treated with 0 or 75 mM NaCl for 2 weeks. Then, the roots and shoots were sampled, dehydrated at 70 °C for 3 days, and weighed. The samples were digested with 65% HNOt 130 °C . The Na+http://geneontology.org/) analysis and p-values were used to assess the significant enrichment of the corresponding category.Four-leave stage rice (MT and WT) was treated with 0 or 100 mM NaCl for 24 h, and then rice roots were harvested to extract the total RNA for the synthesis of cDNA . RNA-seqAdditional file 1.Additional file 2.


Clavaspisselvaticasp. nov., Clavaspisvirolaesp. nov., Davidsonaspistovomitaesp. nov., and Rungaspisneotropicalissp. nov., are described and illustrated from Panama. We also transfer two previously described species of Panamanian Aspidiotini to new genera, Hemiberlesiacrescentiae (Ferris) comb. nov. and Rungaspisrigida (Ferris) comb. nov., and report the first record of Selenaspidopsisbrowni Nakahara in Panama. A key to the species of Aspidiotini occurring in Panama is provided.Four new species of armored scale insect,  Sternorrhyncha, armored scales are strictly phytophagous. Many species of armored scales are pests of agricultural commodities   . The majcaleNet) , but mancaleNet) . Species regions . An iden9.2802°N, 79.9754°W). The locality was chosen because it offered access to the canopy via the crane and because every tree was reliably identified to species. The first survey was conducted in June 2012 by Geoffrey E. Morse and BBN, and the second in January 2015 by G. E. Morse, Daniel A. Peterson, Hannah Shapiro, and Shannon Trujillo. A full description of the sampling protocol is given in 2 bark sample. Collected material was refrigerated and examined under a dissecting microscope within 5 days; live armored scale insects were transferred to 100% ethanol. Subsequently, sampled scale insects were subjected to a joint morphological / molecular sample preparation that resulted in a sample of purified genomic DNA and a permanent microscope slide mount of the specimen’s cuticle, following the method described in The sampling locality for new species described in this paper was the canopy crane in San Lorenzo National Park, Colón . Paratypes: • 1 adult female with second-instar exuviae; same data as holotype; USNM (D6581A); • 2 adult females; same data as holotype; UMEC .N = 4). Adult female, 560–1100 μm wide (holotype 560), broadest at mesothorax or metathorax. Body outline turbinate to nearly oval. Derm membranous throughout at maturity except for pygidium. Antennae simple, each with one long seta. Distance between antennae 100–180 μm. Without disc pores associated with anterior or posterior spiracles. Lobes. L1 well developed, slightly wider than long, inner margins near parallel, with 1 notch on each side or without notches, rounded apically; space between lobes approximately 0.25 times width of L1. L2 and L3 absent. Plates cylindrical, narrow, pointed at apex, simple or with a few fine tines, about as long as L1; 2 plates present in first space, often with 1 or 2 tines near apex giving bifurcate or trifurcate appearance; 1 or 2 plates present in second space, simple or with minute tines; plates absent between L1. Ducts. Dorsal macroducts of 1-barred type, with 2–3 macroducts arising from first space, 8–10 arising from second space, and 7–8 arising from third space in singular rows. Series of marginal macroducts with wide orifices extending from mesothorax to abdominal segment II; at least two present per segment. Groups of ventral submarginal microducts occurring on head, thorax, and abdominal segments I–V. Paraphyses. With 1 pair of paraphysis-like basal scleroses near mesal margins of L1; 1 pair of paraphyses in first space, paraphysis arising from lateral margin of L1 slightly longer than paraphysis arising from medial margin of L2, both mushroom-like in shape with distinctive dome or cap at anterior end; 1 pair of small clavate paraphyses in second space. Anal opening longer than wide, 11–14 μm long, 5–7 μm wide, positioned 17–25 μm  from the base of L1, located within posterior third of pygidium. Perivulvar pores few, 2–6 pores in total, divided into 2–4 groups, with 1–4 in each group.C.coursetiae (Marlatt) with subtle differences distinguishing the two. Submarginal groups of microducts form a semicircle around the head, thorax, and pre-pygidial abdominal segments of C.selvatica but are more diffusely scattered in C.coursetiae, not organized in an obvious semicircular ring. In C.selvatica, at least two large macroducts are present on the mesothorax, while in C.coursetiae only one at most is present, falling near the posterior margin of the mesothorax. The plates are nearly as long as L1 and fringed in C.selvatica but are short and simple in C.coursetiae. This species is also similar to C.subsimilis (Cockerell) in body shape and the shape of L1 but can be distinguished by possessing perivulvar pores on the pygidium (absent in C.subsimilis).This new species is most similar in appearance to Apeibaaspera Aubl. .selvatica is the Latin adjective meaning wild, literally “of the forest” (selva). Our choice of this name is influenced by the fact that in modern Spanish, the word selva is identical to its Latin ancestor in form, but now refers specifically to tropical rainforest.The epithet Panama (Colón).Taxon classificationAnimaliaHemipteraDiaspididaeWei, Schneider, Normark & Normarksp. nov.6755DF26-1264-5232-95C2-BAF943BE77AChttp://zoobank.org/EEB45109-341D-44C4-98B8-E1BEC59F0CB1Holotype: Panama • 1 adult female; Parque Nacional San Lorenzo Canopy Crane, Colón; 9.2802°N, 79.9754°W; 17.i.2015; DA Peterson, GE Morse, H Shapiro, S Trujillo leg.; on Virolamultiflora; MIUP (D6676B). Paratype: • 3 adult females; same data as holotype; USNM ; • 3 adult females; same data as holotype; UMEC .N = 7). Adult female, 410–630 μm wide , broadest near mesothorax and metathorax. Body outline oval, nearly circular in smaller individuals (< 600 μm long), becoming elongate-oval in larger individuals. Derm membranous throughout at maturity except for pygidium. Antennae simple, each with one long seta. Distance between antennae 40–100 μm. Without disc pores associated with anterior or posterior spiracles. Lobes. Pygidium with 2 pairs of lobes; L1 well developed, separated by space about one-fifth width of L1, lobes slightly wider than long, inner margins near parallel, with 1 medial and 1 lateral notch, rounded apically; L2 forming sclerotized point, about one-quarter to one-third size of L1, with 1 lateral notch; L3 absent, indicated at most by small, lightly sclerotized projection of pygidial margin. Plates. All plates simple; with or without fine plates in slight space between L1; with 2 pointed plates in first space; plates absent in second space; five simple microduct-bearing plates present laterad of L3, nearly as long as L1. Ducts. Dorsal macroducts of 1-barred type, slender, with orifices narrower in diameter than ventral microducts, restricted primarily to margin with one submarginal duct anterior to seta marking segment VI; 1 between L1, with 3–4 marginal ducts in first space, 2 marginal ducts in second space; with few short macroducts occurring on submarginal areas of pre-pygidial segments. Ventral microducts slightly wider in diameter than dorsal macroducts and present in small submarginal groups on pre-pygidial abdominal segments and segment V. Paraphyses.L1 each with a paraphysis-like basal sclerosis toward medial margin, slightly smaller than lobe; in first space, 1 clavate paraphysis arising from lateral angle of L1, 1 arising from mesal angle of L2, posterior-most paraphysis slightly longer than L1; 2 smaller clavate paraphyses arising from mesal margin of L3. Anal opening oval, 8–13 μm in length, 4–6 μm in width, positioned 20–23 μm from base of L1, located within posterior third of pygidium. Perivulvar pores absent.Clavaspis MacGillivray on the basis of the robust clavate paraphyses, small anal opening, and basal sclerosis of L1, resembling that of Clavaspisulmi (Johnson). The paraphyses are not as elaborately developed as those of most Clavaspis species, but they are more developed than some species that have recently been recognized as members of Clavaspis on the basis of molecular phylogenetics – C.perseae (Davidson) and C.patagonensis Schneider, Claps, Wei, Normark & Normark , new combination.This species is placed in the genus  Normark . ClavaspVirolamultiflora (Standl.) A.C.Sm. (family Myristicaceae)Virola.The specific epithet is the Latin genitive of the host plant genus, Panama (Colón).Taxon classificationAnimaliaHemipteraDiaspididaeWei, Schneider, Normark & Normarksp. nov.AF12E3F1-DF68-5462-846C-5FF31919BEA1http://zoobank.org/EE712529-F0A9-4EAA-AB50-20F29BB27DF7Holotype: Panama • 1 adult female; Parque Nacional San Lorenzo Canopy Crane, Colón; 9.2802°N, 79.9754°W; 12.vi.2012, GE Morse & BB Normark leg.; on Tovomitalongifolia; MIUP (D3919A). Paratype: Panama • 1 adult female; Parque Nacional San Lorenzo Canopy Crane, Colón; 9.2802°N, 79.9754°W; 15.i.2015; DA Peterson, GE Morse, H Shapiro, S Trujillo leg.; on Tovomitalongifolia; UMEC (D6433A).N = 2). Adult female and narrowly ribbonlike, with minute orifices, few, only 10–15 on each side of pygidium. Also, with faux duct orifice on dorsum immediately anteriad of L1 – circular structure slightly larger than duct orifices, but without duct. Pre-pygidial dorsal macroducts few, shorter than those on pygidium, confined to margin and submargin, absent on segments III and IV, two present on each side of segments II, I, metathorax, and mesothorax. Ventral microducts shorter and thinner than dorsal macroducts, with a few present on submargin of each segment from abdominal segment V to prothorax. Paraphyses. Three pairs of paraphyses present on each side of pygidium, variable, with lateral member of each pair often minute or absent. Medial pair of paraphyses anteriad of L1, medial member of pair arising from near inner angle of L1, extending nearly to anus and terminating in rounded knob, lateral member of pair minute, forming part of sclerotized rim of faux duct orifice; pair of paraphyses between L1 and L2 also with medial paraphysis much larger than lateral paraphysis; pair between L2 and L3 usually about equal to each other in length, lateral member of pair sometimes obsolete. Anal opening nearly circular, maximum diameter 8 μm, located 23–25 μm (about 3 times diameter) from base of L1. Perivulvar pores absent.Davidsonaspistovomitae have been published: the large ribosomal subunit , elongation factor 1-alpha , and carbamoylphosphate synthetase . The small ribosomal subunit (16S) sequences of the primary bacterial endosymbiont, Uzinuradiaspidicola, of the holotype has also been published (KY220578).DNA sequences from 3 loci of the holotype of D.tovomitae has appeared in published phylogenetic trees, where it was labeled “Davidsonaspis ud3919” , found on avocados in Mexico. D.aguacatae had originally been assigned to Abgrallaspis Balachowsky  Hochr. (family Clusiaceae)Tovomita.The specific epithet is the Latin genitive of the host plant genus, Panama (Colón).Taxon classificationAnimaliaHemipteraDiaspididaeWei, Schneider, Normark & Normarksp. nov.5DFB5CD3-8322-5043-9D6C-CA4FF1DF05D6http://zoobank.org/02D416A8-3589-4AC4-877A-3F3A88E7C59BHolotype: Panama • 1 adult female; Parque Nacional San Lorenzo Canopy Crane, Colón; 9.2802°N, 79.9754°W; 20.vi.2012; GE Morse & BB Normark leg.; on Marilalaxiflora Rusby; MIUP (D4168I). Paratypes: • 4 adult females; same data as holotype; USNM ; • 5 adult females; same data as holotype; UMEC .Adult female (N = 10) in some cases pupillarial, enclosed within sclerotized cuticle of 2nd instar; some individuals non-pupillarial. Appearance in life not recorded. Slide-mounted adult female 350–610 μm long , 280–500 μm wide , broadest at mesothorax. Body outline broadly oval, with slight indentation between prothorax and mesothorax. Derm membranous throughout at maturity in pupllarial individuals; cephalothorax and pygidium becoming sclerotized at maturity in some non-pupillarial individuals. Antennae simple, each with one long seta. Distance between antennae 51–73 μm. Eye a submarginal dorsal tubercle on prothorax. Without disc pores associated with anterior or posterior spiracles. Venter of mesothorax with about 6 transverse, irregular rows of sclerotized spicules in submedial area, posterolaterad of mouthparts. Lobes. Pygidium with 1 or 2 pairs of lobes; L1 well developed, subquadrate, with parallel inner margins separated by exceedingly narrow space, lobes slightly longer than wide, rounded apically, with 1 large notch near apex on lateral margin and 0–1 notch near apex on medial margin; L1 each with well-developed basal sclerosis, slightly narrower and longer than lobe; L2, when fully developed, forming a small, sclerotized projection, about one-third length of L1 and much narrower, without notches; L2 often absent or represented by a membranous projection or low, sclerotized point; L3 absent. Plates. With 2 narrow, elongate plates in first space, slightly fringed, with a few tines, and 1 or 2 simple plates laterad of position of L2; no other plates present. Ducts. Dorsal macroducts of 1-barred type, slender, much broader than ventral microducts, few in number, restricted to margin of pygidium; with 1–3  ducts in first space, 0–2  immediately laterad of L2, and 0–1  laterad of seta marking segment VI, making a total of only 1–4 ducts  on each side of pygidium. Ventral microducts exceedingly narrow, present along pygidial margin and scattered in submedial areas of other segments. Paraphyses absent. Anal opening subcircular, 8–11 μm in length and width, positioned 17–37 μm from base of L1, located within posterior half of pygidium. Perivulvar pores absent.Second-instar female (N = 8) Appearance in life not recorded. Slide-mounted second-instar female 340–620 μm long (median 460 μm), 270–400 μm wide (median 340 μm), broadest at mesothorax. Body outline oval. Antennae simple, each with one long seta. Distance between antennae 54–96 μm. Without disc pores associated with anterior or posterior spiracles. Lobes. Pygidium with 3 pairs of well-developed lobes; L1 subquadrate, with parallel inner margins separated by exceedingly narrow space, lobes slightly longer than wide, rounded apically, with 1 large notch near apex on lateral margin and 0–1 notch near apex on medial margin; L1 each with well-developed basal sclerosis, slightly narrower and longer than lobe; L2 nearly as long as L1 but much narrower, rounded at apex, without notches or with slight notch on lateral margin; L3 subtriangular, slightly narrower and shorter than L2, without notches. Plates. Without plates between L1. With 2 narrow plates in first space, 2 broader plates in second space, and a series of 5 or 6 plates laterad of L3. All plates similar in length to adjacent lobes and fringed at apex, with plates anterior to L3 becoming progressively lower and less fringed anteriorly. Plates of the first and second spaces subtended by conspicuous ducts, about a third as wide as dorsal macroducts and nearly as long, much wider and longer than ventral microducts. Ducts. Dorsal macroducts of 1-barred type, broad, all submarginal; with 2 ducts in a short row arising from first space, 2 in the second space, and 1 laterad of L3, making a total of 5 on each side of pygidium. Ventral microducts exceedingly narrow, short, present along pygidial margin and scattered in submedial areas of other segments. Paraphyses absent. Anal opening oval to subcircular, 8–14 μm in length, 7–8 μm in width, positioned 23–40 μm from base of L1, located within posterior half of pygidium.Rungaspisneotropicalis have been published, including fragments of 3 gene regions: the large ribosomal subunit , elongation factor 1-alpha , and cytochrome oxidase I and II .Several DNA sequences of R.neotropicalis have appeared in published analyses and phylogenetic trees, where they were labeled “UG3995 ud3995” , with R.neotropicalis sister to A.fularum. R.neotropicalis was also included in a broader study of Diaspididae , and Entaspidiotuslounsburyi (Marlatt)) but that also includes one other New World species (Rugaspidiotusarizonicus (Cockerell)). It is possible that R.neotropicalis is an African species that is invasive in the Neotropics, similar to Selenaspidusarticulatus, which is the single most abundant diaspidid species at the site where R.neotropicalis was collected , dorsal ducts with sclerotized orifices, and simplified plates located only in the first and second interlobular spaces. African Rungaspis species differ from R.neotropicalis in having conical plates without fringes (vs. slightly fringed) and numerous narrow dorsal ducts (vs. few broad ducts). We considered three other possible placements for the species. One was the genus Aspidiotus Bouché. Rungaspisneotropicalis resembles Aspidiotus species in having basal scleroses of L1 and fringed plates, and molecular evidence indicates that its closest known relative is an African species of Aspidiotus. But we concluded that R.neotropicalis shares a greater number of characters with Rungaspis. Furthermore, Aspidiotus is radically non-monophyletic, and the mostly African clade to which R.neotropicalis belongs should probably be recognized as a distinct genus anyway , a pupillarial species known from South Africa. The two species share a similar overall body shape, L1 with basal scleroses, absence of L3, absence of perivulvar pores, and presence of just a few slightly fringed plates and just a few broad, one-barred dorsal ducts near the pygidial margin. Characters that distinguish R.neotropicalis from A.rhusae are as follows (character of A.rhusae given in parentheses): L2 much narrower than L1 or absent (L2 nearly as broad as L1); space between L1 exceedingly narrow, without plates ; 4 or fewer dorsal ducts present on each side of pygidium (5 or more ducts present); 1–3 microducts present near each posterior spiracle (cluster of 5 or more ducts in this position); transverse rows of minute spicules present on mesothorax posterolaterad of mouthparts (absent); body margin slightly indented between prothorax and mesothorax (entire); eye a submarginal dorsal tubercle . The Neotropical species that R.neotropicalis most closely resembles is Aspidiellarigida Ferris. The two species both have L1 with basal scleroses and closely approximated medial margins, other lobes reduced or absent, cephalothorax becoming sclerotized at full maturity, and perivulvar pores absent. Characters that distinguish Rungaspisneotropicalis from Aspidiellarigida are as follows (character of A.rigida given in parentheses): plates present (absent); dorsal ducts of pygidium broad, much broader than ventral microducts, confined to margin and submargin ; anus in posterior half of pygidium .Morphologically, Rungaspisneotropicalis has further led us to conclude that Aspidiellarigida belongs in the genus Rungaspis, and we transfer it accordingly: Rungaspisrigida (Ferris) comb. nov. Aspidiella. In its pygidial characters it resembles the type genus closely enough except for the entire absence of plates and the absence of the perivulvar pores... In the heavy sclerotization of the entire body it is peculiar and distinctive.” In each of these characters it resembles Rungaspis species more than Aspidiella species. Ferris further expressed puzzlement that an Oriental and Australian genus such as Aspidiella would include a species that was apparently native to the Neotropics. A biogeographic connection between the Neotropics and Afrotropics is better documented  and less of a surprise.Our study of Neotropical and African species that resemble Marilalaxiflora Rusby The specific epithet is a Latin adjective; here it alludes to this species’ unusual biogeography as a Neotropical member of a mostly African clade.Panama (Colón).Taxon classificationAnimaliaHemipteraDiaspididaeNakahara, 1984: 936. New country recordC1CCEC04-2D3E-5AAE-81BF-36378DBB8C3E8.9944°N, 79.5431°W; 22.i.2015; DA Peterson, GE Morse, H Shapiro, S Trujillo leg.; on Antirheatrichantha; MIUP (D6765D); • 1 adult female; same data as previous; UMEC (D6765G).Panama • 1 adult female; Parque Metropolitano Canopy Crane; Antirheatrichantha (Griseb.) Hemsl. (Rubiaceae)Panama (Parque Metropolitano).Hemiberlesiapaucitatis (McKenzie) due to insufficient information.The key incorporates some modified excerpts drawn from Aspidiotini are provided by Additional online resources aiding in the identification of