Study On The Trade Routes Tracing Of The Leiothrix Lutea In China

Leiothrix lutea is a species belongs to leiothrichidae Family,Passeriformes,It is also a traditional ornamental bird in China.it was listed as the national II key protected species in2021.We surveyed 16 bird markets and found illegal trade of Leiothrix lutea still exists in China,which will lead to the decrease of the wild population of Leiothrix lutea;in addition,lack of trade information of Leiothrix lutea restricted the illegal trade control the development of wild bird protection and management measures in China.We used the traditional survey method combined with whole genome re-sequencing technology(SNPs markers)and mitochondrial COI/Cytb markers to track the trade routes of the red billed leiothrix in China,to provide basic information for the wild population protection and illegal trade control of Leiothrix lutea.The results are as follows:1.In 2019,traditional survey methods were used to investigate the wild population distribution and trade status of the Leiothrix lutea in China.The results showed that the trade phenomenon of the red-billed Acacia existed in 16 bird markets in 16 provinces investigated,and the trading individuals in the market were mainly from Yunnan,Guangxi,Sichuan,Guizhou,Shaanxi,Jiangxi.Henan and Anhui,accounting for 33% of the known wild distribution provinces of the Leiothrix lutea in China.The sources of Leiothrix lutea in Shanghai came from Shaanxi,Sichuan and Anhui;trade individuals in Guangdong markets were from Guangxi;Zhejiang and Henan are derived from Henan and Guangxi;Birds in markets of Chongqing,Hubei,Shandong and Jiangsu were from Sichuan.Sichuan is derived from Sichuan and Guizhou;The origin of Gansu is Shaanxi and Sichuan;Anhui derived from Anhui and Guangxi;However,the markets of Guangxi,Jiangxi,Shaanxi,Guizhou and Yunnan are only from local sources.In general,except for Yunnan and Jiangxi,which only found inner-provincial trade,other provinces exsited intra-provincial trade.Moreover,the trade amount are large in Guangxi,Sichuan and Shaanxi.,they were important trade source of the Leiothrix lutea.2.Whole genome resequencing was performed on 141 Leiothrix lutea samples(including 18 wild samples and 123 market samples).After quality control and comparison with the reference genome of the Leiothrix lutea,5 samples with low comparison coverage were discarded,.21,077,599 SNPS markers were obtained from 136 samples.A total of8,231,864 high-quality SNPS markers were used for genetic diversity analysis,principal component analysis(PCA)and genetic STRUCTURE analysis.Genetic diversity analysis showed that the observed heterozygosity(Ho)and expected heterozygosity(He)were0.1045-0.5615 and 0.1026-0.4977,lower than that of Garrulax canorus,G.morrisonianus,Liocichla steerii,Paradoxornis heudei and Buphagus erythrorhynchus.According to the results of PCA and the STRUCTURE,we considered market samples clustered with the wild samples,and the most close one will be more likely the source of trade.No market samples clustered with wild samples from Yunnan and Tibet,indicated the samples were not from Yunnan and Tibet.Trade Leiothrix lutea in Shanghai was from Hubei Province.Trade samples in market of Guangdong was origin from Guangxi and Sichuan;Guangxi was from Guangxi;Zhejiang,Chongqing,Henan,and Gansu were from Sichuan and Shaanxi;Sichuan was origin from Guizhou;Hubei is from Hubei;Jiangxi was from Jiangxi and Hubei;Anhui is derived from Anhui and Sichuan;Shaanxi was derived from Shaanxi;The trade individuals of Guizhou came from Guizhou.The main trade sources of the market samples were Jiangxi,Anhui,Guangxi,Hubei,Sichuan,Guizhou and Shaanxi.3.We directly sequenced the COI gene and Cytb gene of 139 Leiothrix lutea(including 18 wild samples and 121 market samples),and constructed the phylogenetic trees(ML tree and BI tree)by combining the two genes.The results showed that only part of the market samples clustered with the wild samples from known sources.Inferred from the phylogentic tree,Leiothrix lutea trading individual might originate from Guangxi,Hunan,Hubei,Guizhou and Sichuan.Specifically,the trading individuals of Shanghai market originated from Guangxi,Hunan,Sichuan and Guizhou.Trade individuals in market of Guangdong was from Guangxi,Hubei,Hunan,Sichuan and Guizhou.Individuals of Guangxi came from Guangxi,Hunan,Sichuan and Guizhou provinces;Chongqing were originated from Guangxi;Sichuan were derived from Guangxi and Guizhou;Birds in the market of Gansu were from Guangxi and Guizhou;Henan originated from Hubei and Guizhou;Hubei was derived from Guangxi,Sichuan and Guizhou;Jiangxi’s trade individuals came from Hunan;Anhui’s were from Guangxi;Shaanxi was derived from Guangxi,Hunan,Sichuan and Guizhou.Guizhou originated from Guangxi and Guizhou;However,individuals in the bird market of Zhejiang had no clear source by this method.In general,Guangxi,Sichuan and Guizhou were the main trade source of Leiothrix lutea.According to the phylogenetic tree,no individuals in the market were from Yunnan,which was consistent with the results inferred by traditional survey methods and whole-genome resequencing markers.4.STRUCTURE results showed that the genetic structrue was roughly the same with PCA results when the K value was 4,and the wild samples from Yunnan and Tibet were significantly different with those from other regions.The special genetic structure of wild Leiothrix lutea in Tibet and Shaanxi indicated they were more likely the origin of this species,while population of other regions had multiple origins genetically,and there should have been extensive gene flow between them in history.Compared the three methods,there are some defects of the traditional survey as it can’t identify the interference of secondary trade,compared with the whole genome resequencing marker,precision of the mitochondria marker method is low,only less than 50%,it’s difficult to reflect the potential trade routes,meanwhile,the matrilinear inheritance character of the mitochondria make it impossible to trace the origin of hybrid individuals accurately.Therefore,whole-genome resequencing SNP markers was used as the main method for tracing the trade sources and routes of the Red-bill Leiothrix assistanted by the results of the tridational market survey.multiple trade routes were detected: from Guangxi to Guangdong;From Hubei to Shanghai and Jiangxi;From Sichuan to Shanghai,Zhejiang,Chongqing,Henan,Gansu and Anhui;From Shaanxi to Zhejiang,Chongqing,Henan,Gansu and Sichuan;From Guizhou to Sichuan.Sichuan and Shaanxi are the main source of the illegal trade of the Leiothrix lutea,it will be urgent to control the illegal capture of the wild birds in these provinces to protect the wild population of the Leiothrix lutea when fighting against the illegal trade of Leiothrix lutea.