<figure><img src="image_1.png" /></figure>

# Evolutionary history and spread of the *Mycobacterium tuberculosis* Latin American and Mediterranean (L4.3/LAM) sublineage, Tunisia

Mohamed Amine Skhairia, Naira Dekhil *, Helmi Mardassi **

Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

*Mycobacterium tuberculosis*
L4.3/LAM sublineage
Clonal complex
High resolution genotyping
Bayesian estimation
Demographic origin
Evolution
Local adaptation

## ABSTRACT

**Background:** To infer the origin and spread of the *Mycobacterium tuberculosis* Latin American and Mediterranean (L4.3/LAM) sublineage in a Mediterranean country, Tunisia, where it predominates.

**Methods:** We combined Bayesian (STRUCTURE) and maximum likelihood (MIGRAINE) estimation approaches based on a global 24-loci mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR24) genotyping dataset consisting of 1573 L4.3/LAM clinical strains from four continents, including 252 isolates originating from Tunisia.

**Results:** Phylogenetic analyses coupled with Bayesian estimations suggested that the most predominant L4.3/LAM subpopulation in Tunisia (65.07%), which is dominated by a single clonal complex, TUN4.3_CC1 (94.51%), has evolved from an ancestral pool that is restricted to Europe and Africa, contrasting with the remaining L4.3/LAM subpopulations whose ancestry was traced all over the word. Maximum likelihood analysis revealed that TUN4.3_CC1 has been undergoing a demographic expansion since 131 years ago (CI95%: 90.7–205), thus explaining its preponderance relative to the second most predominant CC, TUN4.3_CC2, whose population was found under contraction.

**Conclusions:** The preponderance of L4.3/LAM in Tunisia stems from a 130-year expansion process of a locally evolved clone.

## 1. Introduction

Tuberculosis (TB) is a major infectious disease caused by the *Mycobacterium tuberculosis* complex (MTBC). Genomic data split the latter complex into nine human-adapted lineages, *M. tuberculosis* sensu stricto (L1 to L4 and L7), *M. africanum* (L5 and L6), the recently discovered lineages 8 and 9 that originated in East Africa, and at least nine animal-adapted lineages [1–4]. Among the nine recognized MTBC lineages, L4 is the most geographically dispersed consisting of generalist and specialist sublineages depending on the spread of their ecological niche [5–9]. Genome based phylogeography and comparative genomic analysis have shown that L4 comprises ten sublineages (L4.1.1 to L4.10), with the L4.3/LAM (Latin American & Mediterranean) being the most frequent sublineage, accounting for 20.3% of the dataset [9]. In SIT-VITWEB, a publicly available database, the LAM sublineage proved to be the most frequent, representing approximately 15% of the global TB burden [10,11]. Molecular signature of L4.3/LAM isolates based on 24

variable number of tandem repeats (VNTR) loci identified three global genotypes, RD115, RD174/RD-Rio, and SIT33 [8,10,12–14].

The Latin American-Mediterranean (LAM) family was first identified ~20 years ago based on a phylogenetic analysis of spoligotyping data and was part of a large and heterogeneous Euro-American lineage [15, 16]. The single nucleotide polymorphism (SNP) *fbpC103* in the gene encoding antigen 85 Complex C (Ag85C), and lineage-specific large deletions in regions of difference (RD), RDRio and RD174, have been shown to be associated with higher transmissibility [10,17]. Further, SNP-based phylogeny combined with spoligotype and RD has subsequently identified a number of clades in L4.3 sublineage (L4.3.1 to L4.3.4). As reflected in its name, the L4.3/LAM sublineage appears to have originated in the Mediterranean basin, and then human migrations have led to its global expansion.

The evolutionary history of the L4.3/LAM, in particular, and other LAM sublineages, have been revisited in the post-genomic era, and all the data converge towards the European sourcing with overwhelming

<footnote>* Corresponding author. Unit of Typing and Genetics of Mycobacteria, Institut Pasteur de Tunis, 13 Place Pasteur BP74, 1002, Tunis, Tunisia.</footnote>

<footnote>** Corresponding author. Unit of Typing and Genetics of Mycobacteria, Institut Pasteur de Tunis, 13 Place Pasteur BP74, 1002, Tunis, Tunisia.
E-mail addresses: neiradekhil@yahoo.fr (N. Dekhil), helmi.mardassi@mesrs.tn (H. Mardassi).</footnote>
dispersal to Africa and the Americas [9,18].

In Tunisia, a Maghrebian country, L4.3/LAM strains appear to have descended from two distinct ancestor populations, referred as L4.3_TUN1 and L4.3_TUN2 (65.07% vs 34.92%, respectively) [19]. Overall, the L4.3/LAM population in Tunisia consisted of two main CCs (TUN4.3_CC1 and TUN4.3_CC2), eight minor CCs (TUN4.3_CC3 to TUN4.3_CC10), and 36 singletons. L4.3_TUN1 population comprised TUN4.3_CC1 and TUN4.3_CC6, while TUN4.3_CC2 and the remaining seven CCs were specific to L4.3_TUN2. The two main CCs (TUN4.3_CC1 and TUN4.3_CC2) account for a total of 76.18% (61.5% % and 14.68%, respectively) of the L4.3/LAM population [19]. According to SpolPred [20], TUN4.3_CC1 and TUN4.3_CC2 strains could be linked respectively to L4.3.1 and L4.3.3 as revealed by WGS of 41 representative genomes (Supplementary Table 1). Hence, the success of L4.3/LAM in Tunisia was found to be mainly driven by TUN4.3_CC1, whose local origin has been invoked [19]. Furthermore, TUN4.3_CC1 preserved the RD115 and RD174/RD-Rio regions, suggesting its more ancestral origin relative to the more widespread RDRio genotype.

Here, by combining phylogenetic and Bayesian analysis, using a globally distributed L4.3/LAM 24-loci MIRU-VNTR dataset, including a representative pool from Tunisia, we provide further evidence that the success of L4.3/LAM in Tunisia is the consequence of a long-term expansion of a locally evolved clone.

## 2. Methods

### 2.1. Data collection

To gain insight into the origins and spread of L4.3/LAM isolates prevailing in Tunisia, we compiled a 24-loci mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR24) dataset comprising 1573 globally distributed strains, including 252 L4.3/LAM isolates from Tunisia (Supplementary Table 1).

### 2.2. Population structure analyses

First, the population structure of the global L4.3/LAM dataset was determined using a Bayesian approach implemented in STRUCTURE 2.3.4 software. The parameters of interest were set using a Markov chain Monte Carlo (MCMC) algorithm with a burn-in of $10^5$ iterations and a run length of $10^6$. We used pophelper R package (version 2.3.0) to generate barplot figures and to calculate the delta K using Evanno method [21], with the optimal value of K being deduced using CLUM-PAK server [22].

### 2.3. Phylogenetic inferences

We constructed a minimum spanning tree (MST) based on the MIRU-VNTR24 data using Ridom SeqSphere+ [23]. Clusters were defined as nodes containing strains with identical MIRU-VNTR24 haplotypes, and clonal complexes (CC) as groups with maximum difference of two loci.

### 2.4. Demography dynamics history

Demography dynamics history was explored using the MIGRAINE version 0.6 (http://kimura.univ-montp2.fr/~rousset/Migraine.htm) and a single population model with a single continuous past variation in population size, the bottleneck model OnePopVarSize, as described in Ref. [24]. This model employs Stepwise Mutation Model (SMM) with exponential change in population size starting at time T generations in the past and continuing until the moment of sampling (i.e. present). MIGRAINE does similar analyses as MSVAR [25,26], with a benefit of using of a generalized stepwise mutation model. Four parameters of interest are estimated: ancestral θanc = 2Nancμ, actual θact = 2Nactμ, and D = T/2Nact, where Nact is the current effective population size, Nanc is the ancestral population size (before the demographic change),

and D the time when the demographic change starts, scaled by population size. T is the time measured in generations since the present, and μ is the mutation rate per locus per generation. To convert scaled population sizes (θ and θanc) into effective population sizes (N and Nanc), and scaled times (D) into times in years (T), we used: (1) a generation time of 1 year; (2) mutation rates of $10^{-3}$ mutations per locus per generation for all microsatellite loci [27–29]. The demography dynamics are characterized using the extra parameter Nratio = Nact/Nanc, which is < 1 in the case of a reduction in population size (contraction) and >1 in the case of an expansion. Migraine runs were done using 2000 trees, 500 points and 4 iterations.

## 3. Results

### 3.1. Bayesian population structure analysis of the M. tuberculosis L4.3/LAM sublineage

We carried out Bayesian population structure analyses using MIRU-VNTR24 data of 1573 L4.3/LAM clinical strains recovered from Africa (including the 252 Tunisian L4.3/LAM isolates), Europe, Latin America, and Asia (Supplementary Table S1). As depicted in Fig. 1, the global L4.3/LAM population can be split into six distinct subpopulations (subpopulation 1 to 6). Indeed, multiple runs with K values ranging from 2 to 10 revealed maximal posterior probability at K = 6 (Fig. 1, panels A and B). L4.3/LAM isolates from Africa could be identified among the six divergent subpopulations, particularly in subpopulation 4 (Fig. 1C). Likewise, isolates from Europe are comprised in large numbers in all but one subpopulation, subpopulation 1, which includes essentially isolates from the Americas and a few strains from Africa (subpopulation 1).

The L4.3/LAM clonal complex that predominates in Tunisia, TUN4.3_CC1, fell almost entirely in subpopulation 5, which comprises isolates whose origin is restricted to Europe and Africa (Fig. 1C). On the other hand, TUN4.3_CC2, the second most representative CC in Tunisia, is part of subpopulation 2, which comprises isolates that are distributed throughout the 4 continents (Latin America, Africa, Europe, and Asia). The latter subpopulation also entailed 30.55% of L4.3/LAM Tunisian singletons and the bulk (87.5%) of Tunisian CCs other than TUN4.3_CC1 and TUN4.3_CC2.

### 3.2. Phylogenetic placement of Tunisian L4.3/LAM in a global dataset

The global MIRU-VNTR24-based MST (Fig. 1) showed that European isolates were almost scattered throughout the whole MST of the L4.3/LAM sublineage. Yet, although the majority of isolates from Africa were confined to a single area of the MST, a substantial fraction tended to be represented throughout the whole tree. By contrast, the majority of L4.3/LAM isolates from Latin America grouped together in a distinct branch. Isolates from Asia tended to group together, being split into two distinct subpopulations.

The phylogenetic position of TUN4.3_CC1 and TUN4.3_CC2, which altogether represent the majority of the L4.3/LAM population in Tunisia (76.18%), further confirmed their clear genetic separation. While TUN4.3_CC1 tended to occupy a terminal position of a distinct branch that arose from European and African isolates, TUN4.3_CC2 was linked to a more heterogeneous and larger group comprising isolates from the four continents (Fig. 1 and Supplementary Fig. 1). This result was consistent with a N-J tree from global 12-loci MIRU-VNTR genotyping dataset of 1971 L4.3/LAM clinical isolates from African and European, including 252 isolates originating from Tunisia (Fig. 2). TUN4.3_CC1 proved closely related to L4.3/LAM isolates from the Netherlands, Belgium and Morocco (Fig. 2B), while TUN4.3_CC2 clade proved much more diverse, entailing clinical strains from various origins (Fig. 2C).
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** ANCESTRY analyses and Sub-population assignments of the global L4.3/Latin American and Mediterranean (LAM) dataset (n = 1573) including strains from Tunisia. (A) Each isolate is represented by a single vertical line divided into coloured segments, with length proportional to probability of assignment to each sub-population. (B) Determination of the optimal value of K indicated that the current global L4.3/LAM dataset had descended from 6 distinct ancestor populations. (C) Assignments of L4.3/LAM isolates according to their origin to its corresponding sub-population. (D) Global minimum spanning tree of *Mycobacterium tuberculosis* L4.3/Latin American and Mediterranean (LAM) sublineage based on 24-loci mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR) data. Subpopulation affiliation for each isolate are represented by different colours. The size of a circle is proportional to the number of isolates sharing the same pattern. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

**3.3. Maximum-likelihood-based inference of past changes in L4.3/LAM CCs prevailing in Tunisia**

The population demographic history of the two CCs that make up the bulk of L4.3/LAM in Tunisia, TUN4.3_CC1 and TUN4.3_CC2, was further explored using MIGRAINE under single population models with a single continuous past variation in population size (OnePopVarSize). All analyses were performed using a generalized stepwise mutation model (GSM), which proved robust enough for microsatellite markers, such as MIRU-VNTR and reduces the risk of false positives in bottleneck testing [24,30]. Furthermore, MIRU-VNTRs behave as selectively neutral phylogenetic markers if sufficient numbers of loci are used to buffer against potential biases, and this is why we used data from MIRU-VNTR24.

Using a mutation rates of 10⁻³ mutations per locus per generation for all microsatellite loci, our data indicate that while TUN4.3_CC1 population has witnessed an evolutionary expansion (Nratio = 312) since approximately 131 years ago (CI95%: 90.7–205), TUN4.3_CC2 population has, by contrast, undergone a contraction (Nratio = 0.277) since ~77.2 years (CI95%: 27.8–200) (Table 1, Fig. 3). These results should be treated with caution considering large confidence intervals. Further WGS based studies should help better delineating the past history of L4.3/LAM sublineages in Tunisia.

**4. Discussion**

This study has been prompted by taking into account previous reports suggesting an Eastern Mediterranean or Maghrebian origin for the L4.3/LAM sublineage [31]. These predictions have further been reinforced by uncovering a predominant, long established clonal complex (TUN4.3_CC1), in Tunisia, displaying an intact RD115 region, whose deletion is associated with the global expansion history of L4.3/LAM [8, 18]. Hence, the presence of an intact RD115 region in TUN4.3_CC1 has been interpreted as indicative of its early origin, and it could thus be an extant representative of the progenitor(s) of the L4.3/LAM sublineage. Therefore, to gain further insights into the origin and spread of L4.3/LAM sublineage in Tunisia, we analyzed its phylogeny and ancestry in the context of globally distributed L4.3/LAM sublineage strains. For this purpose, we used MIRU-VNTR genotyping data, which despite the fact that it has less resolution power than whole genome sequencing (WGS) data, it has previously proved to be robust enough to infer the origin, evolutionary history and global spread of *M. tuberculosis* complex strains [32,33]. Furthermore, in order to buffer against potential biases, we used the 24-loci format of MIRU-VNTR typing.

Taken together, the findings presented in this study indicate that the present day success of the L4.3/LAM sublineage in Tunisia, and most likely in the whole Maghreb, stems from the expansion of a single clonal complex, TUN4.3_CC1, whose origin is likely to be indigenous to the African continent. Indeed, phylogenetic and Bayesian analyses further
A

B

C

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Neighbor-Joining tree based on 12-loci mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR) data from 1971 European and African L4.3/LAM isolates. The out-group taxon for tree reconstruction was *M. tuberculosis* H37Rv_Ra. (A) Distribution of TUN4.3_CC1 and TUN4.3_CC2 strains in a NJ tree based on 12-loci MIRU-VNTR from 1971 European and African L4.3/LAM isolates, using *M. tuberculosis* H37Rv_Ra as out-group. Each CC is indicated by its colour code (red for TUN4.3_CC1 and green for TUN4.3_CC2). (B) Clade including TUN4.3_CC1 strains and their closely related European and African isolates. (C) Clade of TUN4.3_CC2 strains and their closely related European and African isolates. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<caption>**Table 1**</caption>

Inferences on demographic history using MIGRAINE expressed by ancestral (N<sub>anc</sub>), current (N<sub>act</sub>) effective population sizes, N<sub>ratio</sub> (N<sub>act</sub>/N<sub>anc</sub>) and time (D<sub>g</sub>) since the demographic change occurred. The 95% confidence intervals are given in brackets.

<table><thead><tr><th>TUN4.3</th><th>Past variation in population size</th><th>Tg<sup>a</sup>mu</th><th>2N<sub>anc</sub>mu</th><th>2N<sub>act</sub>mu</th><th>N<sub>ratio</sub></th><th>Dg<sup>a</sup>mu</th></tr></thead><tbody><tr><td>TUN4.3_CC1</td><td>expansion</td><td>0.001</td><td>0.0209 [0.0139–0.0366]</td><td>6.533 [2.971–15.91]</td><td>312.2 [0.29–2085]</td><td>0.131 [0.0907–0.205]</td></tr><tr><td>TUN4.3_CC2</td><td>contraction</td><td>0.001</td><td>0.765 [0.402–1.535]</td><td>0.212 [0.117–0.436]</td><td>0.277 [0.12–0.919]</td><td>0.0772 [0.0278–0.2]</td></tr></tbody></table>

showed that TUN4.3_CC1 tend to group with strains originating exclusively from Europe or Africa, a finding totally contrasting with the phylogenetic distribution of TUN4.3_CC2, the second most representative CC, which showed phylogenetic links to L4.3/LAM strains distributed throughout the four continents (Latin America, Europe, Africa, and Asia). Hence, we suggest that unlike TUN4.3_CC1, the minor TUN4.3_CC2, arguably, represents the pool of L4.3/LAM dispersed through European colonial migrations that have started in the 16th century [34]. Such a clear genetic separation between the two CCs in a global phylogeny lends further support to our previous WGS-based analysis [19].

Both Bayesian (STRUCTURE) and phylogenetic analyses (MST) inferred an African or European origin for TUN4.3_CC1. This finding is consistent with the very likely African origin of L4 [35]. Because the evolutionary history of TUN4.3_CC1 is clearly distinct from the Out-of-Europe pool, it is tempting to speculate, based on a previously suggested scenario [35], that this successful clone might have been imported to Tunisia, or to a neighboring country, through land routes linking Eastern, Western, and Northern Africa. The fact that phylogenetic links do exist with European strains might be explained by the very past history of the Maghreb, notably the Roman conquest or other following colonial and/or exchange events. On the other hand, it has previously been shown that TUN4.3_CC1 tend to exhibit a higher mean allelic richness compared to TUN4.3_CC2, a finding indicative of its long diversification time [19].

Our data indicate that TUN4.3_CC1 has been spreading in the local population for more than 130 years, thus providing further evidence that it is long-established. Yet, these time estimates should be taken with

caution and have to be challenged by genome-wide nucleotide mutation models. However, such a relatively long expansion period would have contributed to increase its fitness to the local population. Such a hypothesis is supported by clinical findings, since TUN4.3_CC1 expanded more rapidly in the host population than did TUN4.3_CC2 as disclosed by its reduced mean pairwise genetic distance among genomes [19]. Furthermore, given the low rate of drug resistance among TUN4.3_CC1 strains [19], their success is likely to reflect an intrinsic ability to spread rather than a consequence of a compensatory evolution [36,37]. Unlike TUN4.3_CC1, the population size of TUN4.3_CC2 was found to have undergone a contraction. Because TUN4.3_CC2 has likely been introduced into Tunisia through European colonial migrations, its inability to expand in the host population may indicate that it has not yet succeeded in adapting locally [34,38,39].

In sum, our data further confirmed that the clonal complex driving the success of L4.3/LAM sublineage in Tunisia, TUN4.3_CC1, is likely to have emerged locally. Our estimates indicated that this successful clonal complex is undergoing a significant expansion of its population size for more than a century.

## Funding

This study received financial support from the Tunisian Ministry of Higher Education and Scientific Research under grant LR16IPT01.

## Ethical approval

No samples have been processed for the specific need of this study.
TUN4.3_CC1

Profile likelihood ratio

<figure><img src="image_5.png" /></figure>

Profile likelihood ratio

<figure><img src="image_6.png" /></figure>

TUN4.3_CC2

Profile likelihood ratio

<figure><img src="image_7.png" /></figure>

Profile likelihood ratio

<figure><img src="image_8.png" /></figure>

**Fig. 3.** Two-dimensional profile likelihood ratios of the two main CC of L4.3/LAM in Tunisia Migraine runs were done using 2000 trees and the likelihood surface is inferred from a total of 500 points in four iterative steps. The likelihood surface is shown only for parameter combinations that fell within the envelope of parameter points for which likelihoods were estimated. The cross denotes the maximum.

Only fully publicly available, anonymized data, were processed, and hence no further ethical clearance was required.

Data availability statement

General characteristics and genotypic data (24-loci MIRU-VNTR) of the 1573 globally distributed L4.3/LAM strains included in this study are provided in Supplementary Table S1.

Authors' contributions

H. Mardassi conceived and designed this study. M.A. Skhairia, N. Dekhil, and H. Mardassi analyzed and interpreted the data. M.A. Skhairia, N. Dekhil, and H. Mardassi wrote the manuscript. All authors reviewed and approved the manuscript.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.tube.2022.102297.

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