Molecular phylogeny and identification of agromyzid leafminers in China,with a focus on the worldwide genus Liriomyza (Diptera:Agromyzidae)

LIANG Yong-xuan ,DU Su-jie ,ZHONG Yu-jun ,WANG Qi-jing ,ZHOU Qiong ,WAN Fang-haoGUO Jian-yang#,LIU Wan-xue#

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

2 College of Life Sciences,Hunan Normal University,Changsha 410081,P.R.China

Abstract Leaf-mining flies (Diptera: Agromyzidae) are a diverse family of small-bodied insects that feed on living plant tissues as larvae. Various species in this family are considered globally invasive and have caused great agricultural economic losses. In China,economically important vegetable crops have been seriously damaged by these pest insects,especially by species of the genus Liriomyza. However,these species are difficult to differentiate because of their morphological similarities,and the Chinese fauna remains poorly known. To explore the relevant pest species in China and their phylogeny,agromyzid leafminers were collected from 2016 to 2019,and identified based on morphological characteristics and DNA barcodes. In total,27 species from five genera of Agromyzidae were sampled and identified,including 16 species of Liriomyza. Both mitochondrial and nuclear genes were used to reconstruct their phylogenetic relationships and estimate the divergence time. Highly congruent and well-supported phylogenetic trees were obtained using the Bayesian inference and maximum-likelihood methods. This analysis revealed two main clades in Liriomyza,and clade 2 was inferred to have diverged from clade 1 approximately 27.40 million years ago (95% highest posterior density: 23.03–31.52 million years ago) in the Oligocene. Differences were observed in the distribution patterns and host associations between the Liriomyza clades. Clade 2 species are distributed in cool,high-latitude environments,suggesting that they may have evolved into a cool-adapted lineage.

Keywords: agromyzid leafminer,Liriomyza,phylogenetics,identification,divergence time,distribution pattern

1.Introduction

Leaf-mining flies (Diptera: Agromyzidae) are a globally distributed group with phytophagous larvae that feed on living plant tissues,mostly as leafminers,and have been found to attack 140 host plant families worldwide(Spencer 1973,1990;Schefferet al.2007). Adult females cause direct damage when they puncture the leaf surface to feed and lay eggs (Spencer 1973). Larval feeding causes characteristic symptoms on plants,such as leaf mines that vary in appearance,galls,swollen stems,and other symptoms,and injury to the plant,including a reduction in photosynthetic potential and,in some cases,structural damage,leading to declines in plant vigor,growth,and yield (Spencer 1973;Reitzet al.2013).Given their detrimental effect on the agricultural economy,approximately 110 species are considered important pests of cultivated crops worldwide (Dempewolf 2017).

LiriomyzaMik is the largest genus in the subfamily Phytomyzinae afterPhytomyza,with more than 330 described species,24 of which are known to attack agricultural and ornamental plants (Parrella 1987;Kanget al.2009;Reitzet al.2013).Liriomyzaspecies have the widest host plant range and tend to colonize new hosts that are phylogenetically distant from their current hosts(Schefferet al.2007). Most members ofLiriomyzaare monophagous or oligophagous,i.e.,they feed on either a single or a few host species within a family;however,eight species are broadly polyphagous (Spencer 1990).In the past few decades,the occurrence and distribution ofLiriomyzaspecies have changed dramatically (Kanget al.2009;Reitzet al.2013;Gaoet al.2017),including three species that are particularly important pests in most parts of the world,i.e.,L.sativaeBlanchard,L.trifolii(Burgess),andL.huidobrensis(Blanchard) (Murphy and LaSalle 1999;Kanget al.2009;Heet al.2010;Xuet al.2021;Sousaet al.2022).

However,because of their small size and apparent similarity,leaf-mining flies can be difficult to identify,and much of the global fauna remains poorly known.Furthermore,dissection of the male genitalia is essential for the identification of most species (Schefferet al.2007;Lonsdale 2011,2017). Given the difficulties in the morphological identification of leaf-mining flies,molecular markers have become a valuable means for species delimitation and specimen identification (Morganet al.2000;Scheffer and Lewis 2006;Schefferet al.2014;Xuet al.2021;Sousaet al.2022). Using mitochondrial DNA,Morganet al.(2000) and Scheffer and Lewis (2006)revealed high sequence divergence among populations,supporting the presence of cryptic species ofL.trifolii. Xuet al.(2021) and Sousaet al.(2022) used mitochondrial DNA to identify invasiveLiriomyzaspecies in Australia and Brazil,respectively,and to examine their population genetics. Some studies have provided valuable insights into the phylogeny of leaf-mining flies at the family or genus level by using molecular markers (Schefferet al.2007;Winkleret al.2009a,b;Xuanet al.2022). For example,Schefferet al.(2007) conducted a phylogenetic analysis of multiple genera and species in the subfamilies Agromyzinae and Phytomyzinae,and discussed the host-use evolution of species using molecular and morphological data. Winkleret al.(2009a,b) studied the molecular phylogeny of the largest genus,Phytomyza,which has diverse and relatively well-known host associations. They refined the classification of this genus by recognizingNapomyzaandPtochomyzaas subgenera ofPhytomyza,andChromatomyiawas resynonymized with the genusPhytomyza. After completing the diagnosis of fossils of agromyzid leafminers,they calibrated divergence time estimations within thePhytomyzausing the fossil data and provided new insight into the process of adaptive radiation within this genus. Xuanet al.(2022)reconstructed the phylogeny ofLiriomyzausing targetcapture-based phylogenomic datasets and defined groups and clades in this genus using a combination of molecular phylogeny and morphology,which provided a wellsupported phylogenetic framework of this genus.

China has a vast and diversified geographical environment,with various climate types and abundant plant hosts,which is ideal for supporting a diverse agromyzid fauna (Liuet al.2013). The most problematic pestiferous species in China,including the three invasiveLiriomyzaspecies mentioned above as well as the PalearcticP.horticolaGoureau (Fig.1),have caused serious damage to economically important crops,especially vegetables (Kang 1996;Chenet al.2003;Liuet al.2013;Gaoet al.2017). Despite their economic significance in China,few studies have conducted a large-scale sampling and systematic investigation of these species to clarify the diversity and phylogeny of related taxa in the region or provided scientific evidence conducive to their diagnosis,in order to better differentiate the agriculturally important pest species from other species. Chen and Wang (2000) have reported nine species ofLiriomyzaand their distributions in China,but because of the limited diagnostic data,species differentiation remains difficult.

Fig.1 The most problematic pestiferous leaf-mining flies for vegetable and flower crops in China. A1 and A2,Liriomyza sativae,female,from laboratory. B1 and B2,Liriomyza trifolii,male,from Hainan,China. C1 and C2,Liriomyza huidobrensis,female,from laboratory. D1 and D2,Phytomyza horticola,male and female respectively,from laboratory.

Therefore,morphological identification combined with molecular identification and phylogenetic analysis using DNA markers is of great importance in providing a reliable diagnosis of agromyzid leafminer species. The main objectives of the present study were to explore the relevant pest species in China and further investigate their phylogeny. To achieve these objectives,we used a combination of morphological data and DNA barcodes to identify the important agromyzid pests among samples obtained through systematic sampling on common agricultural crops and to generate datasets useful for the diagnosis of those pests. We then constructed phylogenetic trees and estimated the divergence time on the basis of previous studies of Agromyzidae using data derived from both mitochondrial (COI,COII,andCytb) and nuclear (CADand28S) genes. Furthermore,we investigated the distribution patterns and host associations acrossLiriomyzaclades and discussed their divergence and radiation processes. To our knowledge,this is the first comprehensive study that provides a reference for the identification and phylogenetic study of agromyzid leafminers in China.

2.Materials and methods

2.1.Taxonomic sampling

We investigated and collected agromyzid leafminers at approximately 1 000 sites in China from 2016 to 2019(Fig.2). Most sampling sites were greenhouses,vegetable planting stations,farm plots,and community plots. Crop damage was surveyed primarily in the host families Brassicaceae,Fabaceae,Cucurbitaceae,Asteraceae,Solanaceae,and Liliaceae (genusAllium),but also in some others. Sampling date,collection location (longitude and latitude),host plant,temperature,and humidity were recorded (Appendix A). The ArcGIS platform version 10.2 was used to produce the distribution maps. Host plants infested with larvae were collected and transferred to the laboratory,and placed in clean cages until adults emerged. We examined and identified more than 150 000 adult agromyzid leafminers. All adults were preserved in anhydrous ethanol and maintained at–20°C until their use for molecular analysis. Specimens are deposited in the Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing.

Fig.2 Investigation and sampling sites of agromyzid leafminers in China from 2016 to 2019. Each gray dot represents one site,and the red triangles represent sampling sites where the clade 2 species of Liriomyza were found.

Fig.3 Bayesian phylogenetic analysis of Agromyzidae based on partitioned DNA sequences of dataset (i). Posterior probabilities are shown as the colored circles on the corresponding node. The asterisks after a species name indicate that the species were from databases and the literature,while the remaining species were collected in this study. The brackets after the genus Phytomyza indicate that Napomyza and Chromatomyia are a subgenus and a synonym of Phytomyza,respectively (Winkler et al. 2009a,b;Lonsdale and Eiseman 2021).

Fig.4 Maximum likelihood phylogenetic analysis of Agromyzidae based on partitioned DNA sequences of dataset (i). Ultrafast bootstrap support values are shown as the colored circles on the corresponding node. The asterisks after the species name indicate that the species were from databases and the literature,while the remaining species were collected in this study. The brackets after the genus Phytomyza indicate that Napomyza and Chromatomyia are a subgenus and a synonym of Phytomyza,respectively (Winkler et al. 2009a,b;Lonsdale and Eiseman 2021).

2.2.Identification based on morphology

Most species listed in Appendix A were identified based on the comparative morphology of Agromyzidae reported in the literature (Spencer 1973;Lonsdale 2011,2017).Morphological features used for the diagnosis ofLiriomyzaspecies in this study are listed in Appendix B. Specimens ofLiriomyzaspecies were placed on absorbent cotton containing anhydrous ethanol in Petri dishes and observed under Stemi 508 (ZEISS,Oberkochen,Germany) and SZX16 (Olympus,Tokyo,Japan) microscopes. Then,the specimens were transferred onto quartz sand in Petri dishes and photographed using an EOS 750D camera(Canon,Tokyo,Japan) mounted on a BX43 (Olympus,Tokyo,Japan) microscope. Images were rendered and composed using Helicon Focus 6.

2.3.DNA extraction,amplification,and sequence alignment

Whole-body genomic DNA was extracted from individual adult agromyzid leafminers using the rapid grinding method (De Barro and Driver 1997) with minor modifications and the DNeasy Blood and Tissue Kit (Qiagen,Germantown,MD,USA),following the manufacturer’s protocol. The DNA was stored at–20°C until analysis.

Fragments within five genes,i.e.,the mitochondrial genesCOI(cytochromecoxidase subunit I),COII(cytochromecoxidase subunit II),andCytb(cytochromeb) and the nuclear genes28S(28Sribosomal RNA) andCAD(carbamoyl-phosphate synthetase 2,aspartate transcarbamylase and dihydroorotase),were selected as targets. The primer pairs and annealing temperatures used for DNA amplification and sequencing are listed in Appendix C. Polymerase chain reaction (PCR) was performed in a 25-μL reaction mixture comprising 2.5 μL of 10× buffer (including Mg2+),0.5 μL of dNTPs (2.5 mmol L–1),0.4 μL of each forward and reverse primer (10 pmol L–1),0.2 μL ofTaqpolymerase (2.5 U μL–1),1 μL of DNA,and 20 μL of ddH2O. The PCR amplifications were performed under the following thermal conditions: initial pre-denaturation at 94°C for 4 min;35 cycles of 94°C for 30 s,primer annealing at the optimal temperature for 30 s,and 72°C for 90 s;and a final extension at 72°C for 8 min.The PCR products were electrophoresed in 1% agarose gel and stained with a nucleic acid staining solution(GoldView II;Yeasen Biotechnology (Shanghai) Co.,Ltd.,China). Positive PCR products were sent to Sangon Biotech (Shanghai) for sequencing. All DNA sequences obtained have been deposited in GenBank (Appendix A).

We visually inspected and edited the newly generated DNA sequences and aligned them with downloaded related sequences using ClustalW implemented in MEGA 7.0 (Kumaret al.2016). For protein-coding genes(mitochondrial genes andCAD),the sequences were translated into amino-acid sequences using the Expasy Web Portal (https://web.expasy.org/translate/) to verify that they were functional copies.

2.4.Molecular identification and phylogenetic analysis

The primers LCO1490 and HCO2198 were used to amplify theCOIDNA barcodes of collected morphological species (Appendix C). The aligned sequences were analyzed using BOLD (http://www.boldsystems.org/) and BLASTn (Nucleotide Basic Local Alignment Search Tool) searches in the NCBI database(https://blast.ncbi.nlm.nih.gov/) to determine the species or genus of the specimens.

To further study the phylogenetic position of the collected species within Agromyzidae,sequences from other species were downloaded from GenBank to construct phylogenetic trees. In total,63 species(including outgroups) were included in the phylogenetic analysis. Aligned sequences of all species were concatenated using PhyloSuite v1.2.2 (Zhanget al.2020).Sequence accession numbers for the species newly sequenced in this study and reported in previous studies(Appendices A and C) are available in GenBank. Two datasets of aligned DNA sequences were constructed.Dataset (i) includes the mitochondrial and nuclear genes (COI(LCO1490/HCO2198),28S,andCAD) of 63 species,including 61 ingroup species (27 of which were sequenced in this study) and two outgroup species of Agromyzidae (Appendix D). Two species of Odiniidae and Fergusoninidae were selected as outgroups referencing previous studies (McAlpine 1989;Dempewolf 2005;Schefferet al.2007). Dataset (ii) includes the mitochondrial and nuclear genes (COI,COII,Cytb,28S,andCAD) of 19 species,including 16 ingroup species ofLiriomyzaand three outgroup species sequenced in this study (Appendix A).Phytomyzaranunculi,P.vitalbae,andP.horticolaof the subfamily Phytomyzinae were selected as outgroups forLiriomyzain the phylogenetic analysis. Genetic distances amongLiriomyzaspecies were calculated using the Kimura Two-parameter Substitution Model in MEGA 7.0 (Kumaret al.2016),based on the mitochondrial geneCOIand concatenated nuclear genesCADand28Sin dataset (i) (Appendix A),except forLiriomyzasp.4.

We used MODELFINDER 2 (Kalyaanamoorthyet al.2017) and allowed the merging of partitions to select the best-fit partitioning schemes and substitution models(Appendix E) of evolution for both complete datasets based on the Bayesian information criterion. Subsequent phylogenetic analyses were conducted using the Bayesian inference (BI) and maximum-likelihood (ML)approaches. The BI analysis was conducted using the corresponding partitioning scheme in MrBayes 3.2.2(Ronquistet al.2012). We used two parallel runs,which consisted of four Markov Chain Monte Carlo (MCMC)runs for 10 million generations,and the sampling of trees and parameters was set to every 1 000 generations.The first 25% of generations were discarded as burnin and the remaining trees were calculated by posterior probability (PP) using a 50% majority-rule consensus.Convergence of the Bayesian analyses was assessed by confirming that the average standard deviation of the split frequency of two runs fell below 0.01 at completion.For the ML analysis,branch support was estimated by ultrafast bootstrap analysis of 1 000 replicates (Hoanget al.2018). The corresponding partitioning schemes for the 1 000 ultrafast bootstraps (Minhet al.2013) were analyzed using IQ-Tree 1.6.10 (Nguyenet al.2015)to obtain the best ML tree. Branches of the resulting Bayesian tree showing a PP of 0.90–1.0 were regarded as strongly supported,those with a PP of 0.75–0.89 as moderately supported,and those with a PP of 0.50–0.74 as weakly supported. Branches in the resulting ML tree with an ultrafast bootstrap (UFboot) of 90–100% were regarded as strongly supported,those with a UFboot of 75–89% as moderately supported,and those with a UFboot of 50–74% as weakly supported. Trees were visualized using FigTree v1.4.4 (Rambaut 2018).

2.5.Divergence time estimation

The divergence time of the family Agromyzidae was estimated using Beast v.2.6.6 (Bouckaertet al.2014)based on dataset (ii). The best-fitting partitioning scheme and general time-reversible models for nucleotide alignment were the same as those used in the above phylogenetic analyses. An uncorrelated relaxed lognormal clock model was selected,and the substitution rate for theCOIfragment was set to 0.0177,as estimated by Papadopoulouet al.(2010),whereas automatic estimates were selected forCADand28S. A birth-death model was implemented as the tree prior,and an exponential prior was used in the analysis. To calibrate the divergence rate,reported fossil records and a secondary calibration method were used: (1)the stem group age of Agromyzidae was calibrated at 64.4 million years ago (Ma) using the age of agromyzid trace fossils published by Wilfet al.(2006) and Winkleret al.(2009a);and (2) based on a secondary calibration method,a higher-level phylogeny inferred by Winkleret al.(2009a) was used to calibrate the crown group age of thePhytomyzagroup at 39.1 Ma.

The MCMC was run for 200 million generations and trees were sampled every 1 000 generations. We estimated the effective sample size using Tracer 1.7(Rambautet al.2018) to evaluate the convergence of the MCMC. The effective sample size was above 200 for all parameters. The tree files of both runs were resampled 10 000 times and combined in LogCombiner(BEAST package). The maximum clade credibility trees with mean heights as node heights were generated in TreeAnnotator (BEAST package),and the first 25% were removed as burn-in. The mean heights and 95% highest posterior density (HPD) values were displayed using FigTree v1.4.4 (Rambaut 2018).

3.Results

3.1.Data properties

In total,27 agromyzid species from China were distinguished and identified based on morphological and molecular data. These species belong to the generaLiriomyza,Phytomyza,Calycomyza,Ophiomyia,andMelanagromyza(Appendix A). The most represented genus wasLiriomyzacomprising 16 species,and the morphological characteristics of 14 of these species were photographed. Seven important agricultural pest-relatedLiriomyzaspecies were verified. The remaining nine species could not be matched with any known species using BOLD or BLASTn searches in the NCBI database,as they had low sequence identity percentages of 85–93% (Appendix A).

Two concatenated alignment matrices were obtained for phylogenetic analysis. Dataset (i) included 63 species with aligned sequences of 1 672 base pairs (bp) in length,and the data comprised 586 bp ofCOI(LCO1490/HCO2198),646 bp ofCAD,and 440 bp of28S. Attempts to amplifyCADfromP.hirticornis,C.humeralis,andPhytomyzasp.1 were unsuccessful. Dataset (ii) included 19 species with aligned sequences of 3 322 bp in length,including 1 191 bp ofCOI,676 bp ofCOII,373 bp ofCytb,673 bp ofCAD,and 409 bp of28S. We were unable to amplifyCOIandCytbforLiriomyzasp.4. By combining the DNA fragments,we were able to obtain well-resolved BI and ML trees (Figs.3 and 4;Appendices F and G) for phylogenetic analysis and to generate a genetic distance matrix ofLiriomyza(Fig.5;Appendix H).

Fig.5 Heatmap of mean genetic distance values among the species and clades of the genus Liriomyza,based on COI (A) and concatenated CAD and 28S (B) in dataset (i). Species numbered 1–9 belong to clade 1 and those numbered 12–20 belong to clade 2. Darker coloration indicates a higher level of genetic distance,while lighter coloration indicates a lower level of genetic distance. See Appendix H for the genetic distance numeric values.

3.2.Phylogenetic analysis

We constructed BI and ML phylogenetic trees of the family Agromyzidae using dataset (i) (Figs.3 and 4).Both methods yielded highly congruent and well-resolved trees. Agromyzidae species formed a strongly supported group (PP=1.0,UFBoot>90) related to the outgroupsFergusoninaturneriandOdiniasp.(PP>0.9,UFBoot>90).Most species in the family had strong support (PP>0.9,UFBoot>90). Additionally,a strongly supported node(PP=1.0,UFBoot>90) formed two monophyletic clades of the subfamilies Agromyzinae and Phytomyzinae. All genera formed a strongly supported group (PP>0.9,UFBoot>90) on both the BI and ML trees.

Within the subfamily Phytomyzinae,the topologies of the phylogenetic trees of the genusLiriomyzabased on dataset (ii) were consistent with those based on dataset (i) (Figs.3 and 4;Appendices F and G). Within the ingroup ofLiriomyza,most species formed highly supported branches (PP>0.9,UFBoot>90).Liriomyza violivorawas found to be an early diverging species that was a sister to the remainingLiriomyzaspecies,with strong support (PP>0.9,UFBoot>90). Fully supported nodes (PP=1.0,UFBoot=100) formed two distinct clades (clades 1 and 2) on the trees constructed based on both datasets. Clade 1 was divided into three main groups containing as representative speciesL.sativae,L.bryoniae,andL.chinensis,which were marked as “sativaegroup,” “bryoniaegroup,”and “chinensisgroup,” respectively (Figs.3 and 6).Liriomyzasativaewas closely related toL.trifolii,whereasL.huidobrensiswas related toL.bryoniae,with strong support (PP=1.0,UFBoot>90) (Figs.3 and 4;Appendices F and G). Within clade 2,L.baptisiaeandLiriomyzasp.2 were found to be early diverging species.The genetic distance analysis ofLiriomyzabased onCOIand concatenated nuclear genes (CADand28S) (Fig.5;Appendix H) showed that the interspecific mean genetic distances ranged from 0.069 (L.fricki/L.congesta) to 0.210 (L.violivora/L.philadelphivora) and from 0.027(L.fricki/L.congesta) to 0.194 (L.violivora/L.trifolii).Moreover,the mean genetic distances were 0.117 and 0.097 within clade 1,0.099 and 0.057 within clade 2,and 0.141 and 0.152 between the two clades,respectively.

Within the genusPhytomyza,the subgenusNapomyzaformed a sister clade to the remainingPhytomyzaspecies,and the synonymizedChromatomyianested within the main lineage ofPhytomyza(Figs.3 and 4),which is consistent with previous studies (Winkleret al.2009a,b;Lonsdale and Eiseman 2021).Phytomyzahorticola,an important pest in China,was sister toP.syngenesiaewith strong support (PP=1.0,UFBoot>90),and they formed a sister group withP.lactucae,which was strongly supported by the BI tree (PP>0.9).PhytomyzaranunculiandP.vitalbaeformed a sister group with full support(PP=1.0,UFBoot=100),and the group was closely related toP.aquilegiana,as moderately supported by the BI tree (0.75

Within the subfamily Agromyzinae,we found thatOphiomyiasp.1 was placed as sister to an independent lineage of polyphyleticOphiomyia,with strong support(PP>0.9,UFBoot>90).OphiomyiaphaseoliandOphiomyiasp.3 formed a sister group,as moderately supported by the BI tree (0.750.9,UFBoot>90),which is consistent with previous findings (Schefferet al.2007). Within the genusMelanagromyza,Melanagromyzasp.was sister to a group comprisingM.virensandM.minimoides,with moderate support (0.75

3.3.Divergence time estimation

The results of divergence time estimation suggested that the family Agromyzidae originated at approximately 65.56 Ma (95% HPD: 64.40–66.80 Ma),in the early Paleocene (Fig.6). The common ancestor of the genera in the subfamily Phytomyzinae was inferred to have diverged from that of Agromyzinae at 60.66 Ma (95% HPD:54.72–65.84 Ma). Within the Phytomyzinae,Liriomyzawas estimated to have diversified at 37.07 Ma (95%HPD: 31.66–42.73 Ma),whereas the split between the two main clades inLiriomyzawas estimated at 27.40 Ma(95% HPD: 23.03–31.52 Ma) in the Oligocene.Phytomyzawas estimated to have diverged at 39.81 Ma(95% HPD: 39.10–42.23 Ma),with the separation of the subgenusNapomyzafrom the remainingPhytomyzaspecies. Within Agromyzinae,the divergence of the main lineage ofOphiomyiawas estimated at 30.76 Ma (95%HPD: 25.13–37.04 Ma),andMelanagromyzadiverged from the relatedOphiomyiaspecies at 31.44 Ma (95%HPD: 25.86–37.47 Ma).

Fig.6 The chronogram of Agromyzidae estimated in Beast. Numbers at nodes indicate the mean estimated divergence times and blue node bars represent the 95% highest probability density (HPD) intervals. The red circle represents the split between the two clades of Liriomyza. In the geological time scale,Pli indicates Pliocene,and P indicates Pleistocene. Ma,million years ago.

Fig. 7 Geographic distribution records of clade 2 species of Liriomyza. The records of Liriomyza fricki and Liriomyza baptisiae were from Ratnasingham and Hebert (2007) and Lonsdale (2011,2017). The records of species in China were based on the surveys in this study conducted from 2016 to 2019. In addition to the specimens found in this study,some records of Liriomyza congesta were from the Ratnasingham and Hebert (2007).

3.4.Morphological characteristics of Liriomyza

Liriomyza sativae BlanchardMaterial examined. 146♂,198 ♀,CHINA: Hainan,Sanya City,Yacheng Town,18°22′N,109°09′E,February 15,2017,coll. Jing He.

Description. Wing: veins brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 1.6–4.0;vein dm-cu sometimes absent (Lonsdale 2011,2017) (Appendix I),which was also observed inL.sativaepopulations in China. Head: head yellow or deep orange with back of head above foramen,ocellar triangle,clypeus,and posterolateral corner of frons to base of inner vertical bristles dark brown;base of outer vertical bristles in the dark brown area and inner vertical bristles in the border of the yellow and dark brown areas(Appendix I). Thorax: scutum black medially and yellow laterally;katatergite sometimes with posterior margin brown,anatergite dark brown below scutellum and lateral to scutellum with dorsum yellow;pleuron yellow,covered with variable large brown mottling on katepisternum,meron,anepisternum,and anepimeron (Lonsdale 2011,2017);anepimeron sometimes with L-shaped brown mottling (Appendix I). Abdomen: abdomen brown with lateral yellow and tergite 2 often yellow along midline(Appendix I). Legs: legs yellow with coxae brown at base;end,base,or dorsal base of each femur sometimes with brown mottling;tibiae and tarsi dark brown (Appendix I).

Distribution in China. Almost all areas except Tibet.

Liriomyza trifolii (Burgess)Material examined. 38♂,48 ♀,CHINA: Hainan,Sanya City,Tianya District,18°17′N,109°29′E,March 18,2018,coll. Jing He.

Description. Wing: veins light brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 3.0–4.0 (Appendix J). Head: head yellow or deep orange with back of head above foramen,ocellar triangle,clypeus,and posterolateral corner of frons to near base of outer vertical bristles dark brown;base of both inner and outer vertical bristles in the yellow area(Appendix J). Thorax: scutum black medially and yellow laterally;katatergite yellow,anatergite dark brown below scutellum and lighter lateral to scutellum with dorsum yellow;pleuron yellow,covered with large ventral mottling on katepisternum and meron,and anepisternum and anepimeron with small anteroventral spots (Appendix J).Abdomen: abdomen brown with lateral margin yellow;tergites often yellow along midline (tergites 1–2) or with yellow posteromedial emargination (tergites 3–5)(Appendix J). Legs: legs yellow with base of coxae sometimes brown,fore femur with dorsal mottling,base of mid and hind femora sometimes brown dorsally;tibiae and tarsi light brown (Appendix J).

Distribution in China. Mainly in southern areas including Guangdong,Guangxi,Hainan,Fujian,Zhejiang,Jiangsu,Jiangxi,Guizhou,Yunnan,Hunan,and Hubei;also found in a few northern areas such as Liaoning,Shandong,Shaanxi,Hebei,Henan,and Gansu.

Liriomyza huidobrensis (Blanchard)Material examined. 55 ♂,61 ♀,CHINA: Yunnan,Puer City,Lancang Lahu Autonomous County,22°33′23.58′′N,99°56′4.49′′E,April 6,2019,coll. Zhiyong Li.

Description. Wing: veins brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 1.5–2.5 (Lonsdale 2011,2017) (Appendix K).Head: head yellow with back of head,ocellar triangle,clypeus,and posterolateral corner of frons to base of inner vertical bristles dark brown;base of outer vertical bristles in the dark brown area,and inner vertical bristles often in the border of the yellow and dark brown areas(Appendix K). Thorax: scutum black medially and yellow laterally;katatergite brownish,anatergite dark brown below and lateral to scutellum with dorsum yellow;pleuron yellow,covered with variable large brown mottling on katepisternum,meron,anepisternum,and anepimeron(Lonsdale 2011,2017) (Appendix K). Abdomen: abdomen dark brown,sometimes yellowish emargination medially on tergite 2 (Appendix K). Legs: legs yellow with dark brown mottling on base of coxae and dorsal of femora;tibiae and tarsus dark brown (Appendix K).

Distribution in China. Most areas,except Guangdong,Hainan,Hunan,Jiangxi,Jiangsu,Shandong,Liaoning,and Heilongjiang.

Liriomyza bryoniae (Kaltenbach)Material examined.6 ♂,8 ♀,CHINA: Shanxi,Linfen City,Hongdong County,36°30′32.52′′N,111°41′33.03′′E,July 19,2019,coll.Xuan Wang.

Description. Wing: veins light brown,yellowish near base;length ratio of final section to penultimate section of vein CuA1: 1.5–2.5 (Appendix L). Head: head yellow with back of head above foramen,ocellar triangle,clypeus,and posterolateral corner of frons to near base of outer vertical bristles dark brown;base of both inner and outer vertical bristles in the yellowish area (Appendix L). Thorax: scutum black medially and yellow laterally;katatergite yellowish,anatergite dark brown below scutellum and yellowish lateral to scutellum with dorsum yellowish;pleuron yellowish,covered with large ventral mottling on katepisternum and meron,and anepisternum and anepimeron with small anteroventral spots that are often lighter than those ofL.trifolii(Appendix L).Abdomen: abdomen brown with lateral margin yellowish;the brown areas of posterior margins of tergites 2–6 slightly curved;brown mottling on tergite 2 of abdomen yellow along midline,and both parts of mottling sloped(Appendix L). Legs: legs yellowish with base of coxae sometimes light brown;femora often yellowish;tibiae and tarsi light brown (Appendix L).

Distribution in China. Mainly in northern areas such as Beijing,Hebei,Henan,Shanxi,Gansu,Inner Mongolia,Ningxia,and Xinjiang.

Liriomyza brassicae (Riley)Material examined.5 ♂,6 ♀,CHINA: Yunnan,Xishuangbanna Dai Autonomous Prefecture,Mengla County,21°56′1.37′′N,101°14′35.86′′E,April 7,2018,coll. Sujie Du.

Description. Wing: veins brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: usually 2.5–3.5 (Lonsdale 2011,2017) (Appendix M). Head: head yellow or deep orange with back of head above foramen,ocellar triangle,clypeus,and posterolateral corner of frons to base of vertical bristles dark brown;base of outer vertical bristles in the dark brown area,and inner vertical bristles in the border of the yellow and dark brown areas (sometimes base of inner vertical bristles in the yellow area) (Appendix M). Thorax:similar to the corresponding part ofL.sativae;mottling on anepimeron sometimes detached (Appendix M).Abdomen: abdomen brown with lateral margin yellow;tergites sometimes with thin medial dividing yellow line(tergite 2) or posterior margin yellow (tergites 3–5),tergite 6 sometimes with large brown medial spot (Appendix M).Legs: legs yellow with base of coxae light brown;base or dorsal base of femora sometimes light brown;tibiae and tarsi brown (Appendix M).

Distribution in China. Only found in Yunnan in this study.

Liriomyza chinensis (Kato)Material examined. 16 ♂,17 ♀,CHINA: Shandong,Tai’an City,Taishan District,36°11′7.95′′N,117°20′46.15′′E,September 27,2019,coll. Baohua Ye.

Description. Wing: overall light color relative to other species,veins light brown,yellowish near base;length ratio of final section to penultimate section of vein CuA1:3.0–4.0 (Appendix N). Head: head yellow with back of head above foramen,ocellar triangle,clypeus and the base of outer vertical bristles brown,posterolateral corner of frons to near base of outer vertical bristles dark brown;base of outer and inner vertical bristles in the brown and yellow area,respectively (Appendix N). Thorax:scutum grey-black medially and yellow laterally,scutellum entirely grey-black with no luster;katatergite dark brown,anatergite dark brown below scutellum and yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large brown mottling on katepisternum,meron,anepisternum,and anepimeron (Appendix N). Abdomen:abdomen dark brown,sometimes yellow emargination medially on tergite 2 (Appendix N). Legs: legs yellow with coxae light brown at base sometimes;femora yellow;tibiae and tarsi light brown (Appendix N).

Distribution in China. Mainly in northern areas such as Beijing,Hebei,Henan,Shandong,Shanxi,Gansu,Inner Mongolia,Jilin,and Heilongjiang.

Liriomyza sp.1Material examined. 19 ♂,28 ♀,CHINA:Beijing,Daxing District,39°36′13.10′′N,116°18′35.08′′E,May 20,2019,coll. Jing He and Meng Guo;9 ♂,14 ♀,CHINA: Beijing,Changping District,Ming Tombs Town,40°14′20.34′′N,116°12′5.57′′E,May 20,2019,coll.Liting Pan;7 ♂,11 ♀,CHINA: Shandong,Dezhou City,Lingcheng District,37°29′2.36′′N,116°32′2.12′′E,May 17,2019,coll. Jing He and Huihui Chen.

Description. Wing: veins brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 3.0–4.0 (Appendix O). Head: head yellow with back of head above foramen,ocellar triangle and clypeus brown;posterolateral corner of frons yellowish;base of both inner and outer vertical bristles in the yellow area (Appendix O). Thorax: scutum black medially and yellow laterally;katatergite yellowish,anatergite dark brown below scutellum and yellowish lateral to scutellum with dorsum yellow;pleuron yellowish,covered with large ventral mottling on katepisternum and meron,and with small and light (sometimes very faint to indistinct)anteroventral spots on anepisternum and anepimeron(Appendix O). Abdomen: abdomen brown with lateral margin yellowish,tergites often yellowish along midline (tergites 1–2) or with yellowish posteromedial emargination (tergites 3–5) (Appendix O). Legs: legs yellowish with coxae and femora often yellowish;tibiae and tarsi light brown (Appendix O).

Distribution in China. Beijing and Shandong.

Liriomyza sp.2Material examined. 7 ♂,CHINA:Jilin,Baicheng City,Taobei District,45°33′58.35′′N,122°49′51.39′′E,June 6,2018,coll. Cuiting Liu and Yao Fu.

Description. Wing: veins brown,yellow near base;length of ultimate section of vein CuA1divided by penultimate section: 3.0–4.0 (Appendix P). Head:head yellow with back of head above foramen,ocellar triangle and clypeus brown;posterolateral corner of frons yellowish;and base of both inner and outer vertical bristles in the yellow area (Appendix P). Thorax: scutum black medially and yellow laterally,shape of black area characteristically different from that in other species;katatergite yellowish,anatergite dark brown below scutellum and yellowish lateral to scutellum with dorsum yellow;pleuron yellowish,covered with large ventral mottling on katepisternum and meron;anepisternum and anepimeron without anteroventral spots (Appendix P).Abdomen: abdomen brown with lateral margin yellowish,tergites often yellowish along midline (tergite 2) (Appendix P).Legs: legs yellowish with coxae yellowish and light brown mottling on dorsal of fore femora sometimes;tibiae and tarsi light brown (Appendix P).

Distribution in China. Jilin.

Liriomyza sp.3Material examined. 8 ♂,CHINA:Heilongjiang,Harbin City,Daowai District,45°47′23.13′′N,126°42′34.67′′E,July 5,2018,coll. Sujie Du.

Description. Wing: veins brown,yellow near base;length of ultimate section of vein CuA1divided by penultimate section: 3.0–4.0 (Appendix Q). Head: head yellow with back of head above foramen,ocellar triangle,clypeus and posterolateral corner of frons to base of outer vertical bristles dark brown;base of both inner and outer vertical bristles in the yellow area (Appendix Q). Thorax:scutum black medially and yellow laterally;katatergite yellow,anatergite dark brown below scutellum and yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large brown mottling on katepisternum and meron,small spots on anepisternum and anepimeron,the spot on anepimeron is half-moon shaped (Appendix Q).Abdomen: abdomen brown with lateral yellow and tergite 2 often yellow along midline (Appendix Q). Leg: legs yellow with base of coxae dark brown;end,base or dorsal base of femora often with brown mottling;tibiae and tarsi dark brown (Appendix Q).

Distribution in China. Heilongjiang.

Liriomyza sp.4Material examined. 3 ♂,5 ♀,CHINA:Gansu,Jiuquan City,Dunhuang County,40°07′1.40′′N,94°37′51.38′′E,April 28,2019,coll. Yujun Zhong.

Description. Wing: overall light color relative to other species,veins light brown,yellowish near base;vein dmcu absented in all individuals (Appendix R). Head: head light yellow with back of head above foramen,ocellar triangle and clypeus brown;posterolateral corner of frons yellow,and base of both inner and outer vertical bristles in the yellow area (Appendix R). Thorax: scutum dark brown medially and yellow laterally with brown mottling on left and right sides,shape of black area differentiated from that in other species;katatergite yellow,anatergite dark brown below scutellum and yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large ventral mottling on katepisternum and meron,and with small anteroventral spots on anepisternum and anepimeron(Appendix R). Abdomen: abdomen brown with lateral margin yellow,tergite sometimes yellow along midline(tergite 2) and with yellow posteromedial emargination(tergites 3–5) (Appendix R). Leg: legs yellow with base of coxae brown;base and dorsal of femora often with brown spots;tibiae and tarsi brown (Appendix R).

Distribution in China. Gansu.

Liriomyza sp.5Material examined. 2 ♂,CHINA: Inner Mongolia,Alxa leagu,38°50′53.62′′N,105°41′36.36′′E,September 6,2019,coll. Jing He and Huihui Chen.

Description. Wing: veins light brown,yellowish near base;length ratio of final section to penultimate section of vein CuA1: 3.0–4.0 (Appendix S). Head: head yellow or yellow with back of head above foramen,ocellar triangle and clypeus brown;the posterolateral corner of frons yellow,and base of both inner and outer vertical bristles in the yellow area (Appendix S). Thorax: scutum and scutellum similar toLiriomyzasp.4;katatergite yellow,anatergite dark brown below scutellum and yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large ventral mottling on katepisternum and meron,anepimeron with small anteroventral spots,anepisternum sometimes without any spots (Appendix S). Abdomen:abdomen brown with lateral margin yellow,tergites 1–5 with thick yellow area along midline (Appendix S). Leg:legs yellow with coxae and femora entirely yellow;tibiae and tarsi light brown or dark yellow (Appendix S).

Distribution in China. Inner Mongolia and Gansu.

Liriomyza sp.6Material examined. 5 ♂,CHINA:Jilin,Baicheng City,Taobei District,45°33′58.35′′N,122°49′51.39′′E,June 6,2018,coll. Cuiting Liu and Yao Fu.

Description. Wing: veins brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 3.0–4.0 (Appendix T). Head: head yellow with back of head above foramen,ocellar triangle and clypeus brown;the posterolateral corner of frons yellow,and base of both inner and outer vertical bristles in the yellow area (Appendix T). Thorax: scutum dark brown medially and yellow laterally with special shape dark brown area;katatergite yellow,anatergite dark brown below scutellum and yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large ventral mottling on katepisternum and meron,while anepisternum and anepimeron with lighter anteroventral spots which is similar toL.trifolii(Appendix T). Abdomen: abdomen brown with lateral margin yellow and tergite 2 yellow along midline (Appendix T). Leg: legs yellow with tibiae,tarsi and base of coxae light brown;light brown mottling on dorsal of femora sometimes (Appendix T).

Distribution in China. Jilin.

Liriomyza sp.7Material examined. 2 ♂,2 ♀,CHINA:Heilongjiang,Mudanjiang City,Yangming District,44°35′19.59′′N,129°41′31.94′′E,September 8,2019,coll. Hong Wang.

Description. Wing: veins light brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 2.5–3.5 (Appendix U). Head: head yellow or orange with back of head above foramen,ocellar triangle and clypeus brown,the posterolateral corner of frons yellow;base of both inner and outer vertical bristles in the yellow area (Appendix U). Thorax: scutum black medially and yellow laterally;katatergite yellow;anatergite dark brown ventral to scutellum and yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large ventral mottling on katepisternum and meron,anepisternum and anepimeron with light and small anteroventral spots(Appendix U). Abdomen: abdomen brown (tergite 1 only with dorsally small brown spot) with lateral margin yellow,tergites 1–2 often yellow along midline and tergites 3–5 with yellow posteromedial emargination (Appendix U).Leg: legs yellow with coxae brown at base sometimes;brown mottling on dorsal of femora;tibiae and tarsi dark brown (Appendix U).

Distribution in China. Heilongjiang.

Liriomyza sp.8Material examined. 6 ♂,6 ♀,CHINA:Jilin,Baicheng City,Taobei District,45°33′58.35′′N,122°49′51.39′′E,June 6,2018,coll. Cuiting Liu and Yao Fu.

Description. Wing: veins brown,yellow near base;length ratio of final section to penultimate section of vein CuA1: 3.0–4.0 (Appendix V). Head: head yellow with back of head above foramen,ocellar triangle and clypeus brown,the posterolateral corner of frons yellow;base of both inner and outer vertical bristles in the yellow area(base of outer vertical bristles sometime in the border of yellow and brown areas) (Appendix V). Thorax: scutum black medially and yellow laterally;katatergite yellow;anatergite dark brown ventral to scutellum and half yellow lateral to scutellum with dorsum yellow;pleuron yellow,covered with large ventral mottling on katepisternum and meron,anepisternum and anepimeron with small anteroventral spots which is similar toL.trifolii(Appendix V). Abdomen: abdomen brown with lateral margin yellow,tergite 2 sometimes yellow along midline (Appendix V).Leg: legs yellow with coxae brown at base;brown mottling on dorsal and base of femora;tibiae and tarsi brown(Appendix V).

Distribution in China. Jilin.

4.Discussion

4.1.Phylogeny of Agromyzidae

We derived the phylogenetic relationships of Agromyzidae,including the species collected in China,from wellresolved phylogenetic trees (Figs.3 and 4;Appendices F and G). The genera involved in this study were from the subfamilies Phytomyzinae and Agromyzinae,and the phylogenetic relationships among the genera based on our analysis were consistent with previous studies (Schefferet al.2007). Moreover,we found that the generaLiriomyza,Phytomyza,Calycomyza,andMelanagromyzawere monophyletic groups,whereas the genusOphiomyiaremained polyphyletic (Schefferet al.2007;Winkleret al.2009a,b;Xuanet al.2022).

The origin of the family Agromyzidae was estimated at approximately 65.56 Ma (95% HPD),in the early Paleocene (Fig.6),which is consistent with the oldest(64.4 Ma) known fossil record of Agromyzidae from the early Paleocene in southeastern Montana (USA) (Wilfet al.2006;Winkleret al.2009a,2010). This estimation is also close to the time (70.26 Ma) when Agromyzidae diverged from Fegusoninidae as estimated by Zhaoet al.(2013) based on mitochondrial genomes. The genusPhytomyzadiversified at approximately 39.81 Ma (95%HPD: 39.10–42.23 Ma),which is in line with previous findings (Winkleret al.2009a). Some species diverged relatively recently. For example,the split between the two related speciesP.hirticornisandP.lateraliswas estimated at 0.86 Ma (95% HPD: 0.31–1.53 Ma),andP.horticoladiverged fromP.syngenesiaeat 2.75 Ma(95% HPD: 1.68–3.94 Ma).Phytomyzaranunculidiverged fromP.vitalbaeat 5.45 Ma (95% HPD: 3.60–7.51 Ma),which is consistent with a Pliocene fossil ofP.ranunculirecorded in Germany (Straus 1967,1977)(Fig.6).

Global climate variation will directly affect the distribution,abundance,and diversity of plant species,and thus indirectly affect the species formation and evolution of related herbivore species (Nymanet al.2012). Some studies have suggested that leaf-mining,as the most specialized and main feeding type,may have been especially affected by the Cretaceous-Paleogene mass extinction event,in which numerous insect herbivores became extinct (Labandeiraet al.2002). The origin and early evolution of Agromyzidae may have been closely related to that event. The unbalanced food webs and changes in climate and plant composition in the early Paleocene after the extinction event may have provided opportunities for the evolution and expansion of agromyzid leafminers (Winkleret al.2010).

4.2.Distribution patterns and host associations across the Liriomyza clades

The combined results of the phylogenetic analysis and the field investigation indicate that the interspecific relationships followed an obvious geographic pattern,as species that occurred in similar latitude ranges had close phylogenetic relationships (Table 1;Figs.3–5 and 7;Appendices F and G). This was especially true for species in clade 2,which showed smaller genetic distances,similar distribution patterns,and cool-adapted plant-host associations,suggesting that they may be more closely related than those in clade 1 in the phylogeny.

Most species in clade 1,such as the invasive speciesL.sativae,L.trifolii,L.huidobrensis,L.bryoniae,andL.brassicae,are generally considered worldwide pests.They are widely distributed throughout continents and countries in the tropics,temperate zones,and even cold zones,and continue to spread to other regions (Spencer 1973,1990;Parrella 1987;Kang 1996;Murphy and LaSalle 1999;Weintraubet al.2017;Xuet al.2021). In addition,they are reported to be widely distributed in China (Kang 1996,2009;Liuet al.2013),which is in line with our findings from the field surveys. The sites where species in clade 2 were collected in China are shown in Table 1,Appendix A and Fig.2. They were only distributed in areas at high latitudes in the north temperate zone with a low average temperature,including the Shanxi,Gansu,Jilin,and Heilongjiang provinces. Among these species,L.congestawas also recorded in Canada (Table 1;Fig.7)(Ratnasingham and Hebert 2007). The two other clade 2 species,L.frickiandL.baptisiae,were also recorded in high-latitude regions and even northern frigid zones of North America (Table 1;Fig.7),including Alaska (USA)and the Yukon Territory and Northwest Territories (Canada)(Lonsdale 2011,2017).

We observed obvious differences in host plants species between the two clades (Table 1). Within clade 1,the five invasive species mentioned above were polyphagous,with a wide host plant family range,including Asteraceae,Fabaceae,Brassicaceae,Cucurbitaceae,Solanaceae,and Malvaceae (data not shown),which is consistent with the findings in previous studies (Spencer 1990;Kang 1996;Kanget al.2009;Liuet al.2013;Xuanet al.2022). Clade 1 also included some oligophagous and monophagous species,such asL.trifoliearumandL.chinensis(Tranet al.2007;Xuanet al.2022). However,the species in clade 2 were found to mainly use relatively cool-adapted plants as hosts in China,such as species in the families Asteraceae and Fabaceae (Pisumsativum) (Table 1).LiriomyzafrickiandL.baptisiaewere reported to use genera within the family Fabaceae as host plants in previous studies(Lonsdale 2011,2017).

Table 1 Geographic distribution and host plants recorded for Liriomyza philadelphivora and clade 2 species of Liriomyza

4.3.Phylogeny of the Liriomyza clades

The phylogenetic relationships amongLiriomyzaspecies based on the BI and ML trees were well-resolved. However,some unknown species collected in China also failed to match the species recently published by Xuanet al.(2022)through molecular comparison. Based on the phylogenetic trees,L.violivorawas an early diverging species of the genusLiriomyza,in line with previous findings (Schefferet al.2007;Xuanet al.2022). Moreover,we found two distinct clades (clades 1 and 2),which corresponded to some of the representative clades in previous studies,and the topologies of both clades were not different from them(Schefferet al.2007;Xuanet al.2022). For example,within clade 1,thesativae,bryoniae,andchinensisgroups correspond to thepusilla/trifolii,strigata,andmoriogroups defined by Xuanet al.(2022),respectively,whereas the branch ofL.brassicaecorresponded to theirbrassicaegroup.Liriomyzasativaewas closely related toL.trifolii,consistent with previous studies (Schefferet al.2006,2007;Carapelliet al.2018;Chenet al.2019;Xuanet al.2022),whereasL.langeiwas found to be the most closely related species toL.huidobrensis(Xuanet al.2022).According to the positions ofL.baptisiae,L.fricki,andL.congestaon the phylogenetic trees,clade 2 probably corresponds to thehieraciigroup defined by Xuanet al.(2022),which means that the species in thehieraciigroup may share a clade with the species recorded in China in this study. Interestingly,the species in thehieraciigroup,such asL.ptarmicae,L.orilliensisandL.minor,were also reported to be distributed in high-latitude regions with cool climates in Europe or North America (Xuanet al.2022;Ratnasingham and Hebert 2007).

Studies have shown that climate change and host plants are important factors affecting the evolutionary patterns of leaf-mining flies (Schefferet al.2007;Winkleret al.2009a,b). For instance,Phytomyzaspecies quickly exploited the ecological opportunities provided by the climate-driven expansion of an asterid-dominated herbaceous vegetation in the Miocene through repeated host transfer. After the cooling events in the middle Miocene climatic transition (MMCT,13.9 Ma),with the rapid expansion of cool and open habitats,the abundance and diversity of herbaceous asterid groups in the north temperate regions increased,and the main radiations of asterid-feeding groups followed almost immediately(Winkleret al.2009a). Evolution driven by climate and host plants has been verified in studies of other taxa(Graham 1996;Vojeet al.2009;Nymanet al.2012;Liet al.2020;López-Lópezet al.2021;Zhuet al.2022).Given the good adaptability of the clade 2 species ofLiriomyzato cool environments (Table 1;Fig.7),some paleoclimatic events leading to climate cooling,such as the MMCT and the earliest Oligocene glacial maximum(33.5 Ma),may also have provided clade 2 species with ecological opportunities for adaptive radiation,and driven their evolution into a lineage adapted to cool climates and cool-adapted host plants (Zachoset al.2001;Holbournet al.2005;Katzet al.2008). The timing of these two events is consistent with the splitting and diversification of clade 2,respectively.

Like many other genera,Liriomyzais a highly specialized genus. Broadly polyphagous species with a wide host range and spanning multiple plant classes are very rare. Only eight polyphagous species have been reported (Spencer 1990;Ward and Spaulding 1993;Xuanet al.2022),includingL.sativae,L.trifolii,L.huidobrensis,L.bryoniae,andL.brassicae,which were detected in clade 1 in this study. However,the actual topology of clade 1 is complex and the number of species in this clade is large (Xuanet al.2022). The polyphagous species did not form a single monophyletic group,but rather represented distinct lineages within this clade on the trees,which was consistent with previous study(Xuanet al.2022). In addition to polyphagous species,clade 1 contained oligophagous and monophagous species,such asL.chinensisandL.trifoliearum,as well as some other species not detected in this study,such asL.eupatoriella,L.citreifemorataandL.lupini(Xuanet al.2022). Given the differences in the distributions and host plant associations of the existing species in clade 1(Spencer 1973,1990;Parrella 1987;Kang 1996;Murphy and LaSalle 1999;Tranet al.2007;Xuanet al.2022),there may be substantial differences in the phylogenetic processes between these two clades,and even among the species within this clade.

5.Conclusion

This study investigated the agromyzid leafminer species collected through systematic sampling on common agricultural crops in China. In total,27 species from five genera were differentiated,as supported by identification and well-supported phylogenetic trees,which provides useful morphological characteristics and molecular data for the diagnosis of agromyzid leafminers in China. The analysis revealed that two main clades had formed in the important agricultural-related genusLiriomyza,with distinct differences in distribution patterns and host associations between the two clades,which may have followed distinct phylogenetic processes. Our results represent a valuable resource for further study of the taxonomy and molecular phylogeny of this group.

Acknowledgements

This study was supported by the National Key R&D Program of China (2021YFC2600400 and 2022YFC2601100),the National Natural Science Foundation of China (31772236 and 31972344),and the Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (caascx-2022–2025-IAS).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.04.030