A novel Ancestral Beijing sublineage of Mycobacterium tuberculosis suggests the transition site to Modern Beijing sublineages

Global Mycobacterium tuberculosis population comprises 7 major lineages. The Beijing strains, particularly the ones classified as Modern groups, have been found worldwide, frequently associated with drug resistance, younger ages, outbreaks and appear to be expanding. Here, we report analysis of whole genome sequences of 1170 M. tuberculosis isolates together with their patient profiles. Our samples belonged to Lineage 1–4 (L1–L4) with those of L1 and L2 being equally dominant. Phylogenetic analysis revealed several new or rare sublineages. Differential associations between sublineages of M. tuberculosis and patient profiles, including ages, ethnicity, HIV (human immunodeficiency virus) infection and drug resistance were demonstrated. The Ancestral Beijing strains and some sublineages of L4 were associated with ethnic minorities while L1 was more common in Thais. L2.2.1.Ancestral 4 surprisingly had a mutation that is typical of the Modern Beijing sublineages and was common in Akha and Lahu tribes who have migrated from Southern China in the last century. This may indicate that the evolutionary transition from the Ancestral to Modern Beijing sublineages might be gradual and occur in Southern China, where the presence of multiple ethnic groups might have allowed for the circulations of various co-evolving sublineages which ultimately lead to the emergence of the Modern Beijing strains.

or the Beijing strain as defined by spoligotyping, is composed of several sublineages broadly categorized into the Ancestral and Modern Beijing strains 8 . The Ancestral Beijing strains forms a phylogenetic cluster that exhibit a cascading feature with the Modern Beijing strains forming a clade at the end of the cascade. The latter has a star-shaped phylogeny, caused several outbreaks around the world and is associated with drug resistance in some countries 5,9 . They are common and appear to be expanding in many countries 5 and, therefore, have been extensively studied. Their frequently used genetic markers are mutations in two genes related to DNA repair and recombination, namely mutT2 codon 58 (G1286766C, mutT2-58) and ogt codon 12 (C1477596T, ogt12) 10 as well as the insertion of IS6110 (insertion sequence 6110) in the NTF (noise transfer function) genomic region 8 . Other identifiers have also been used, such as a set of 44 SNPs (single nucleotide polymorphisms) 11 .
To understand their spreading potential, the geographical origin of L2 or the Beijing strains are sought and debated. A number of studies suggested that they started in Southern China 7,12 and subsequently spread throughout Eastern Asia, from Tibet 13 to Japan 14,15 . The Modern Beijing strains appeared to successfully co-expand with Han Chinese in Northern China 7 . It is unclear however whether the Modern Beijing strains originated there or in Southern China, moved with people and subsequently expanded in Northern China 7 . An alternate hypothesis is that the Beijing strains originated in Northern China 16,17 as the Ancestral strains are common in Japan 14,15 and Korea 18 as well as in Jilin province in Northern China 12 .
Here we report the WGS analysis of M. tuberculosis isolates from a cohort of 1170 patients in Chiangrai province, Northern Thailand during 2003-2010. Some lineages and sublineages exhibited clear association with particular ethnicity of patients. Among the L2 strains, the Ancestral Beijing strains were associated with ethnic minorities. We discovered 40 isolates, which had only the mutT2-58 mutation, considered as typical of the Modern Beijing strains. This sublineage was more common among Akha and Lahu ethnic minorities than Thai patients. Both tribes speak the Lolo-Burmese division of the Tibeto-Burman language family 19 . They were originally from Southern China and moved to northern Thailand only in the last 100 years 20 . We hypothesize that this sublineage descended from an ancestor that was an intermediate between the Ancestral and the Modern Beijing strains and, therefore, suggested that the evolutionary transition from the former to the latter occurred in Southern China.

Results
phylogenetic analysis of M. tuberculosis in chiangrai. 1170 successfully sequenced M. tuberculosis isolates were classified by phylogeny into Lineage 1-4 (L1-4) based on phylogenetic analysis of SNVs (single nucleotide variants) identified across their entire genomes. The results were completely consistent with the classification by experimental LSP (large sequence polymorphism). In total, 70937 SNVs were identified, with 33527 SNVs (47%) present in only a single isolate. The Bayesian and maximum likelihood phylogenies are shown in Figs 1 and S1 respectively. The number of isolates in each lineage was 480 (41.0%) for L1, 521 (44.5%) for L2, 11 (0.94%) for L3 and 158 (13.5%) for L4. The number of isolates belonging to each sublineage as well as some of their genetic descriptions are shown in Supplementary Table S1.  www.nature.com/scientificreports www.nature.com/scientificreports/ The classification of L1 isolates was previously reported 3 . The isolates belonging to L2 were classified based on the phylogenies shown in Figs 1 and S1A-C, and designated using the schemes proposed by Shitikov et al. 21 and Mestre et al. 10 sequentially. The details of the classification criteria are shown in Supplementary Table S1.
The prevalence of L2.1 was 2.3% of L2 isolates, which was similar to the prevalence reported from Chiba prefecture in Japan (2.35%) 22 but lower than that reported from Guangxi (6.1%) 12 Supplementary Table S1. The majority (64 and 164 isolates) belonged to the Asia Ancestral 2 and 3 sublineages respectively. Nine and six isolates fit the descriptions of Bmyc6 and Bmyc26 respectively 21 . Each of them was not monophyletic, however, and were therefore considered as unclassified. The last sublineage, comprising 40 isolates, unexpectedly had one of the barcoding SNPs of the Modern sublineages, G1286766C (mutT2 codon 58) but not the ogt12 mutation. This sublineage did not have the 44 SNPs proposed to be specific to the Modern Beijing strains 11 and most of them did not have IS6110 in the NTF region either. Five of the isolates had a copy of IS6110 inserted in the NTF region, at the position 3493756 and in the opposite orientation with respect to the one typically found in the Modern Beijing strains. This new sublineage, designated as L2.2.1.Ancestral 4, had a relative small average intragroup pairwise SNV distance of 127.4 and a high fixation index of 0.629, as shown in Supplementary Table S1, as well as a clear separation from other Ancestral Beijing strains in a principal component analysis (PCA) plot shown in Supplementary Fig. S2C. A single isolate with only mutT2-58 mutation was previously reported from a patient in Shanghai 11 . As shown in Fig. 1 Supplementary Fig. S3, which reveals that the Fujian isolate was closer to the root of tree and thus suggests that L2.2.1.Ancestral 4 in Chiangrai diverged after the isolate.
The 235 Modern Beijing isolates could be identified into six major groups. The numbers of isolates belonging to the L2.2.1.1 (Pacific RD150) and the Asian African 3 sublineage were 22 (9.3% of Modern Beijing strains) and 29 (12%) respectively. 70 (29.5%) isolates had the SNPs A2376135G and G2532616A, indicating that they belonged to either the Asian African 2 sublineage or L2.2.1.2 (Asian African 2/RD142) with only two of them having the RD142 deletion and, therefore, belonging to L2.2.1.2. 21 isolates (8.9%) had a mutation, C720902T, previously described as a marker specific to a single isolate from Thailand, designated as Bmyc22 10 . Unlike the cases of Bmyc6 and Bmyc26, all Bmyc22 isolates were monophyletic. A single isolate could be classified as Bmyc20 10 . The other 92 Modern strains did not fit to any previously described subsets of L2.2.1 and were referred to here as unclassified Modern Beijing isolates.
There were only 11 L3 isolates in this study. All cannot be subclassified based on Coll's scheme 26 . 158 L4 isolates diversified into three major branches as shown in Supplementary Fig. S1D-E, which could be further classified 26 into 6 sublineages, corresponding to 4.1-4.5 and L4.8. The majority of the L4 isolates belonged to L4.5 (93 isolates, 58.9%) and L4.4.2 (37 isolates, 23.4%). Both were similarly found in high frequencies in China 27 . The L4.5 strains did not include any isolates with SIT127 or related strains, which were recently described as L4.5.1/Iran 28 . L4.5 strains were tentatively classified into two subgroups, namely L4.5.2 and L4.5.3, based on their relatively large between group mean pairwise SNV distances (391.1) and fixation indices of 0.304 and 0.268 respectively, as well as the PCA plot shown in Supplementary Fig. S2D.
Spoligotypes. The spoligotypes of 480 L1 isolates were previously described 3 . The spoligotypes of the L2-4 isolates are shown in Supplementary Table S3A-C respectively.
The most common L4 spoligotypes (46.2%) were 777777777760771 (SIT53), found in four of the six sublineages. All other spoligotypes of L4 could be derived from SIT53 by deletion, suggesting that the most recent common ancestor (MRCA) of L4 had the spoligotype of SIT53. Spoligotypic clades of L4 were not congruent with the SNP phylogeny either. Nevertheless, T2 clade belonged to L4.4 or L4.2 but not L4. 5 TOTAL  patients  480  521  265  64  146  40  235  22  68  29  21  93  11  158  38  74  19  1170   Sex: Male  354  357  176  42  103  24  166  16  47  21  17  63  9  110  23  49  18  830   Female  126  164  89  22  43  16  69  6  21  8  4  30  2  48  15  25  1 , was significantly more common among Thais while the L2 (p = 8.803 × 10 −11 , χ 2 (1) = 42.07) and L4 (p = 3.287 × 10 −10 , χ 2 (1) = 39.496) lineages were significantly more common among non-Thai patients. The higher prevalence among non-Thai individuals was found only for the L2.2.1.Ancestral sublineages but not for the L2.2.1.Modern sublineages as indicated by the risk ratios (RR) in Supplementary Table S4. Most patients infected with L2.1 were Thai, but the number of samples were small. Among specific ethnic minority groups, the distributions of sublineages among the linguistically and genetically related Akha and Lahu, were similar but there were differential associations with L2. In general, the clinical profiles of patients infected with various sublineages of L1 were fairly similar. However, the patient profiles among sublineages of L2 varied significantly, in particular the Modern sublineages. For example, isolates belonging to L2.2.1.1 (Pacific RD150) were not found in any non-Thai patients. while all the other sublineages could be found as shown in Supplementary Table S4. The Asian African 3 sublineage had high drug resistance rates while the rates of Bmyc22 were very low. The patients infected by the latter tended to be young (average age = 33.9 ± 10.8 years) but had a high rate of HIV infection. The variations between sublineages may explain the variations in the results from previous phenotypic association studies of the Beijing strains 31 , which could be due to the differences in sublineage compositions.   Supplementary Table S5. Mutations in 13 genes were predicted to affect their protein functions. An interesting function-affecting mutation was found in an essential gene, aftD, encoding arabinofuranosyltransferase enzyme. This enzyme is involved in the synthesis of the arabinan domain of major mycobacterial cell envelope glycolipids, arabinogalactan and lipoarabinomannan 32,33 . The SNP may affect cell wall functions and isoniazid activity 34 .
Other notable mutated genes include uvrD1, which plays a role in the repair of multiple forms of DNA damage, including site-specific chromosomal double-strand breaks. Ablation of UvrD1 functions sensitizes the bacteria to ionizing radiation 35,36 . However, the Asian African 3 specific SNP in uvrD1 was not predicted to affect its function even though a minor effect could not be ruled out. UvrD1 plays a role in persistence of M. tuberculosis infection in a murine model 37,38 .
Other virulence-related genes that harbour function-affecting SNPs specific for the Asian African 3 strains include fadD29 responsible for phenolic glycolipid (PGL-tb) biosynthesis 39 , cstA, carbon starvation protein A homolog, mce1A, related to bacterial cell entry to host cells 40 and Rv0140, a reactivation-associated antigen 41 .

Discussion
The population structure of M. tuberculosis in Chiangrai is highly complex, comprising L1-L4, and each lineage, except for L3, could be classified further into several sublineages. This might be results of the complex history of Chiangrai which has been a settlement since 7 th century and alternately controlled by several tribes now residing in Thailand, Myanmar and Lao. It is also settled by several hill tribes and more recently by Chinese immigrants. www.nature.com/scientificreports www.nature.com/scientificreports/ This study revealed several interesting features of the population structure of M. tuberculosis, which have a number of implications.
First, Chiangrai was unusual in that the Ancestral Beijing strains were more common than the Modern Beijing strains, which was different from China in the north 27 and Bangkok in the south 23 . The Ancestral Beijing strains were associated with ethnic minorities, who mostly migrated from Southern China through Northern Myanmar, Lao and Vietnam. These areas were, therefore, likely to have high prevalence of Ancestral Beijing strains as well.
We previously compared country-wide proportions of Beijing isolates and the isolates with single-banded IS6110 RFLP, majority of which belonging to Lineage 1. The proportion of Beijing strains was higher among younger patients and generally decreased with distances from Bangkok, the capital of Thailand, while the reverse was true for the single-banded isolates 9 . Lineage 1 was hypothesized to be endemic in Thailand before the emergence of the Beijing strains, presumably by various immigration waves of Han Chinese mostly via marine routes through Bangkok. The discovery of a large proportion of the Ancestral Beijing sublineages and their association with ethnic minorities who originally migrated from Southern China, indicates the significance of the land route in the movement of Ancestral Beijing sublineages as well.
Second, there were a considerable number of isolates genetically similar to rare or belonging to unknown genotypes, such as L2.2.1.Bmyc22 10 , the unclassified Modern Beijing sublineages and surprisingly L2.2.1.Ancestral 4. This study thus demonstrated the need for careful phylogenetic analysis in WGS studies of M. tuberculosis. The finding that different bacterial genotypes were associated with different ethnic groups suggested a possibility of encountering new or rare genotypes when bacteria circulating in a new population are to be studied.
The third interesting finding was the variations of demographic and clinical profiles among different sublineages, particularly among L2, which might reflect different activities of transmission. For example, the very low intragroup average pairwise SNV distance of the Bmyc22 (49.8), together with the low average age and high HIV co-infection rate suggested a relatively high recent transmission activity. This knowledge may be useful for developing public health control of the spreading sublineages. Differential drug resistance rates were also observed, especially among the Modern Beijing sublineages. The classification of Beijing strains only broadly into the Ancestral and the Modern strains may not be adequate for indepth genotype-phenotype association studies, as the results may change 11,31 if the compositions of sublineages vary. With the increasing availability of WGS data of M. tuberculosis in the near future, an internationally agreed guideline for genotypic classification of M. tuberculosis is needed. The high resistance rates among some sublineages also highlight the need for drug susceptibility studies in controlling tuberculosis. It still needs to be investigated whether the drug resistance variability contributes to the difficulty in controlling tuberculosis in some areas or not.
The fourth finding is the discovery of L2.2.1.Ancestral 4 sublineage, which harbored both mutT4-48 and mutT2-58 but not ogt12 mutations. This indicated that the identification of the Modern Beijing strains by barcoding SNPs of mutT2-58 and ogt12 mutations 10 , needs revision probably by including more SNPs from the set of 41 SNPs specific to the Modern strains. It also posed some doubts on the hypothesis that the MutT2−58 together with the MutT4-48 mutations might provide some evolutionary advantages of rapid adaptation for the Modern Beijing strains, by increasing their mutation rates, because both mutations were found in L2.2.1.Ancestral 4 and the ogt12 mutation was silent 10,42 . The selective advantages may be explained by the 41 specific SNPs. Alternatively, the mutations in the putative mutator genes by themselves might decrease the fitness of the strains, which required compensatory mutations 42 existing only in the Modern Beijing strains or the apparent advantages of the Modern Beijing strains may be actually contributed by some sublineages, and not the entire group of Modern sublineages. In any case, it was clear that mutT2-58 could not be reliably used as a SNP marker for identifying the Modern Beijing strains.
The fact that a considerable number of L2.2.1.Ancestral 4 existed in Chiangrai and was much more prevalent among the two linguistically and genetically related Akha and Lahu tribes 43 than the native Thai population illustrates the phylo-ethno-geography of M. tuberculosis in a local and regional scale. As Akha and Lahu were originally from Yunnan or Southwestern China 20 , the sublineage might be originally present there. An isolate in Fujian which appeared to branch out before the isolates in Chiangrai conformed to this hypothesis. The sublineage might have adapted and become endemic to Akha and Lahu but was subsequently out-competed by the Modern Beijing sublineages in most parts of China. Alternatively, both tribes, which dwell in overlapping areas, might have acquired the sublineage along the southward migratory route, such as in Eastern Myanmar. More information about the population structures of M. tuberculosis in Myanmar or Lao are required to further examine this hypothesis. Nevertheless, it is however unlikely that Akha and Lahu acquired and adapted to the sublineage in Thailand because they only recently arrived at Northern Thailand about 100 years ago. Moreover, at first they dwelled almost exclusively in the mountainous areas separated from other ethnic groups. It was only after 1980s with several development programs that they had become integrated economically and then socially with native Thais on the plain of Chiangrai. Recently there has also been some slow but continuous physical migration waves of some hill tribes, particularly Akha to urban areas of Chiangrai. We hypothesized that along with these events, L2.2.1. Ancestral4 and L4.5.2, which might be previously more exclusive among hill-tribes have been spilling over to Thais and other ethnic populations.
The possible origin of L2.2.1.Ancestral 4 in Southern China was supported by the high prevalence of L4.5.2 among Akha and Lahu as well. L4.5 was previously described as a specialist L4 sublineage 44 , due to its geographic restriction mainly only to China. The phylogeny suggested the separation of L4.5 into two subgroups, L4.5.2 and L4.5.3, which were distinct by their spoligotypes, drug resistance rates and the associations to Akha and Lahu. The associations were strong and significant only for L4.5.2. L4.5 has been proposed to have separated from other sublineages of L4 for at least a millennium and may have spread from Tibet 28 to other places in China. This study suggested that L4.5.2 might have accompanied Tibeto-Burman tribes who migrated southward as well. In this case, the association of the sublineage with ethnicity in Chiangrai may be mainly due to the founder effect, although some contribution of co-evolution might also exist. www.nature.com/scientificreports www.nature.com/scientificreports/ The presence of the Ancestral 4 sublineage in the ethnic groups originally from Southern China suggested that the mutations that transformed the Ancestral sublineages may have been acquired gradually before finally reaching the state of the Modern Beijing strains. Some of these changes occurred in Southern China probably in the areas historically occupied by Akha and Lahu ethnic groups. It should be noted that, currently, both tribes are scattered across a mountainous area, extending from the Himalayan mountains toward the South China Sea and covering Southern China, Northern Myanmar, Thailand, Laos and Vietnam as illustrated in Fig. 4. This area was also populated by numerous groups of other ethnic minorities or hill tribes, speaking several hundred different languages belonging to various language families, including the Tibeto-Burman, Tai-Kadai, Hmong-Mien and Austroasiatic. The area is composed of mountainous ranges that run mostly from the temperate north to the tropical south alternating with deep valleys containing river plains. The vast diversity of altitude and latitude create diverse climate and environments, allowing for vast biodiversity 45 , where various ethnic groups can thrive separately in different habitats. The high language diversity suggests a high genetic diversity among ethnic minority populations. The areas are remote due to harsh mountainous terrain, allowing many tribes to live in their traditional ways and probably maintain their genetic segregation. Many areas are not yet covered well with modern health services and hence not studied. It is possible that there are more variants of the Beijing strains, some of which may have partial characteristics of the modern strains, circulating in the areas. Each may have been maintained by co-evolution with specific ethnic groups. www.nature.com/scientificreports www.nature.com/scientificreports/ This hypothesis, together with the high prevalence of L2.1 in Guangxi, conforms to the hypothesis that the origin of the Lineage 2, L2.1 and probably L2.2 or the Beijing strains was in Southern China 7 , where L2.2.2 or the RD181-positive Beijing strains were also highly prevalent 12 . The early L2.2 or Ancestral Beijing strains spread widely from Tibet 13 to Japan 14,15 . In south Eastern Asia they may have diversified by co-evolving with various genetically diverse non-Han Chinese ethnic tribes in the segregated mountainous areas. The emerging sublineages fail or thrive for millennia, resulting in the present variety of the Ancestral Beijing lineages. Some of them presumably gained novel mutations, making them more and more similar to the present Modern sublineages. They might have been subsequently transmitted to the ancestors of Han Chinese, as illustrated in Fig. 4. As the latter population expanded in Northern China, the Modern Beijing sublineages also co-expanded to become predominant strains throughout China and East Asia as found presently 8 . Further studies in the area would provide more insight to the evolution of L2 or the Beijing strains, allow us to understand more about the evolution of pathogenesis of in these microbes and hopefully allow the design of a better tuberculosis control tool. Chiangrai is an area with ancient settlement and has become a major tourist attraction site and a transportation hub to Myanmar, Lao and China through the Mekong River. The recent economic growth of Chiangrai has resulted in substantial migration of populations from other parts of Thailand as well as from neighboring countries. The population of Chiangrai is about 1.2 million with the tuberculosis incidence rate of 152.6/100,000 population in 2011. Most of the populations were native Thais who may be genetically admixed with Chinese and others. Ethnic minorities constitute a considerable proportion of Chiangrai populations. Most of them live in villages in mountainous areas and are known collectively as hill tribes 20 . There were six major tribes based on their spoken languages. Akha, Lahu, Karen and Lisu speaks languages belonging to the Tibeto-Burman family while Hmong and Iu-Mien (Yao) languages belong to the Hmong-Mien family 46 . The ethnicity of the patients was recorded as self-identified.
Akha and Lahu speak the Lolo-Burmese division of Tibeto-Burman language family 19 . They probably started to establish in Yunnan 2000 years ago from the Tibet region. They migrated through Myanmar and arrived at Northern Thailand about 100-200 years ago 20 . ethics Statement. The project was approved by the Ethical Committees of Chiangrai Prachanukroh Hospital, Chiangrai and the Ministry of Public Health, Thailand. Informed consent was obtained from all participants and/or their legal guardians. All methods were performed in accordance with the relevant guidelines and regulations.
Bacteria. Bacterial samples from 1187 patients were recultured in Lowenstein-Jensen medium in an appropriately contained clinical microbiology laboratory in Chiangrai using standard biosafety protocols and equipment. DNA was extracted as previously described 3 . All the processes were performed in Class II biosafety cabinets.
Bacterial genotyping. LSP and spoligotyping of all isolates were experimentally determined as previously Whole Genome Sequencing and SNV analysis. The samples were sequenced on the Illumina HiSeq 2000 platform to produce paired end reads, at the Sanger Institute, UK and processed as previously described 3 . The number of available reads with acceptable scores of five isolates were too small for further analysis. Twelve samples appeared to contain mixed nucleotide sequences and were also not further studied. The sequencing data of samples used in this paper were submitted to the European Nucleotide Archive (ENA) of EMBL-EBI which are mirrored in the Sequence Read Archive (SRA) database. Actual read sequences can be queried and downloaded directly from the SRA database using the study accession numbers ERP006738 and submission accession numbers ERA398050, ERA407418, ERA411689, ERA414376 and ERA428771. phylogenetic analysis. The remaining SNVs from 1170 isolates were used for phylogenetic tree reconstruction with Maximum Likelihood (ML) and Bayesian Inference (BI) methods using PHYLIP 47 , PhyML 48 and MrBayes 49 , respectively. The ML trees were supported by 1,000 replicates of pseudosamples, and the BI tree was Analysis of phenotypic data. The patient characteristics were described in descriptive statistics as presented in Table 1 and Supplementary Table S4. The associations between phenotypes and the four lineages were evaluated by one-tailed Pearson Chi-square test. The P-values less than 0.05 were considered as significant.
Associations with ethnicity were analyzed by categorizing patients based on self-identified ethnicity regardless of nationality. The risk ratio (RR) of each ethnic group was calculated compared to the Thai patients. The RR of all other Tai-Kadai speaking tribes including Lao were calculated together due to the small sample size. The RR for the Tibeto-Burman were presented for Akha and Lahu separately and all the others (which included Burman) combined. The Hmong-Mien group was composed of Hmong and Yao (Iu-Mien).
The HIV and drug resistance rates were calculated based on samples for which the data were available. The MDR rates were calculated based on the number of samples that both susceptibility to INH and RMP was known.
Statistical analysis. Categorical variables of clinical profiles, such as sex, ethnicity, clinical presentation, HIV status, drug resistance were presented as number. Continuous variables were presented as the mean and standard deviation (SD) and the median and interquartile range (IQR). The chi-square test or Fisher's exact test and Yates correction were applied in the analysis in this study as appropriate. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using R version 3.4.3 (R Foundation).
Analysis of non-synonymous L2.2.1.Asian African 3 sublineage specific SNPs (LS-SNPs). SNPs specific to L2.2.1.Asian African 3 were identified with the non-synonymous ones listed in Supplementary  Table S5. The effects of the nonsynonymous LS-SNPs on protein function were predicted by two algorithms, Polyphen-1 and SIFT, from online consensus classifier PredictSNP1.0 53 . The nsSNPs influencing protein function were identified in 7 functional categories.

Data Availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.