PhD Maximilien Vermandel
email: maximilien.vermandel@chru-lille.fr

MD Mathilde Quidet

PhD Anne-Sophie Vignion-Dewalle

MD Henri-Arthur Leroy

Bertrand Leroux

PhD Serge Mordon

MD, PhD Nicolas Reyns

Florent Auger

Comparison of different treatment schemes in 5-ALA interstitial photodynamic therapy for high-grade glioma in a preclinical model: An MRI study

Keywords: 5-ALA, PpIX, Photodynamic Therapy, Magnetic Resonance Imaging, PDT, MRI, Glioblastoma, High-Grade Glioma

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background

There is currently no therapy that prevents high-grade glioma recurrence. Thus, these primary brain tumors have unfavorable outcomes. Recently, 5-ALA photodynamic therapy (PDT) has been proposed to delay relapse and is highly expected to have potential synergistic effects with the current standard of care. However, PDT treatment delivery needs to be optimized by evaluating the impact of both the number of fractions and the light power used.

Objectives

Previous studies have reported MRI examination-based outcomes for PDT in glioblastoma.

Our study aimed to compare MRI markers across different treatment schemes that use interstitial PDT in high-grade glioma in a preclinical model.

Materials and Methods

Forty-eight "nude" rats were grafted with human U87 cells into the right putamen and subsequently submitted to interstitial PDT. The rats were randomized into six groups, including two different sham groups and four different treated groups (5 fractions at 5 mW or 30 mW and 2 fractions at 5 mW or 30 mW). After photosensitizer (PS) precursor (5-ALA) intake, an optical fiber was introduced into the tumor. Treatment effects were assessed with early high-field MRI to acquire T1 and T2 diffusion and perfusion images.

Results

There was no difference in the variation of the diffusion coefficient among the six groups (p=0.0549, Kruskal-Wallis test). However, a significant difference was identified among the six groups in terms of variation in perfusion (p=0.048, Kruskal-Wallis test), supporting a lesional effect in the treated groups. Additionally, the sham groups had significantly smaller edema volumes than were observed in the treated groups. Moreover, the 5-fraction group treated with 30 mW was associated with edema volumes that were significantly greater than those in the 5-fraction group treated with 5 mW (p=0.019).

Conclusion

Based on observations of MRI data and considering treatment effects, the 5-fraction group treated at 5 mW was not significantly different from the other treated groups in terms of cell deaths, characterized by diffusion imaging, or necrosis level. However, the significantly lower level of edema observed in this group indicated that this treatment scheme had limited toxicity.

I. Introduction

Glioblastoma (GBM) is a malignant brain tumor with a particularly poor prognosis and a median overall survival of approximately 15 months given the current standard of care (SOC) [START_REF] Iacob | Current data and strategy in glioblastoma multiforme[END_REF][START_REF] Ostrom | The epidemiology of glioma in adults: a "state of the science" review[END_REF][START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. Although GBM is a rare neoplastic disease with a low prevalence (0.3/10,000 persons), it remains the most frequent primary malignant brain tumor in adults [START_REF] Grill | Glioblastoma[END_REF][START_REF] Fisher | Epidemiology of brain tumors[END_REF].

Currently, the European Society of Medical Oncology (ESMO) guidelines provide recommended treatment options according to the grade of the glioma [START_REF] Stupp | Highgrade glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF]. Given the invasive nature of GBM, local relapse is a universal occurrence and is the reason that surgery is followed by radiotherapy (RT) to the tumor bed and concomitant and adjuvant temozolomide chemotherapy (TMZ), which prolong both progression-free survival (PFS) and overall survival (OS). Although adjuvant treatment after surgery significantly improves results, its benefits remain modest (less than 3 months over the median OS with RT+TMZ versus RT alone (3)). Therefore, the management of newly diagnosed patients should include maximal resection when achievable. The extent of resection (EOR) has prognostic value.

Currently, no existing therapeutic agent is available that can stop GBM progression, and complete resection is rarely feasible because tumor cells usually infiltrate the surrounding brain tissue. Adjuvant therapies to improve local control are thus highly expected.

Recently, 5 amino levulinic acid (5-ALA) photodynamic therapy (PDT) was proposed to be delivered intraoperatively during GBM resection within the INDYGO clinical trial 1 . Although this trial aims to investigate the safety and feasibility of 5-ALA PDT during neurosurgery procedures, it is highly expected to have potential synergistic effects with SOC in the patients enrolled in the trial who harbor newly diagnosed GBM.

The concept underlying 5-ALA PDT relies on the specific accumulation of the photosensitizer protoporphyrin IX (PpIX) in GBM cells because they lack several enzymatic cascades, which usually leads to heme biosynthesis. After PpIX is excited with a 635 nm laser light for 5-ALA PDT, free radical production and oxidative species formation [START_REF] Henderson | How does photodynamic therapy work?[END_REF] lead to cytotoxic reactions within GBM cells. The presence of oxygen is thus required, and treatment delivery should involve tissue oxygenation to maximize therapeutic effects [START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF]. Alternating "on" and "off" periods of light (i.e., fractionation) might improve tissue reoxygenation during the "off" periods that follow oxygen consumption during the "on" periods to address this issue [START_REF] Curnow | Light dose fractionation to enhance photodynamic therapy using 5-aminolevulinic acid in the normal rat colon[END_REF].

In a previous study, we used 2-fraction and 5-fraction schemes to deliver the same light dose of 25 J, and we demonstrated that light fractionation during interstitial PDT (iPDT) enhanced therapeutic effects [START_REF] Leroy | Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model[END_REF][START_REF] Tetard | Light Dose Fractionation to Enhance Photodynamic Therapy With 5-Aminolevulinic Acid in Human glioma xenografts[END_REF]. These effects were assessed in a preclinical GBM model using both histopathological analyses and non-invasive techniques that relied on MRI imaging [START_REF] Leroy | MRI assessment of treatment delivery for interstitial photodynamic therapy of high-grade glioma in a preclinical model[END_REF]. 5-ALA PDT induced targeted tumor lesions, and its primary effects included necrosis, apoptosis, and reduced central tumor vascularization, as confirmed by immunohistology. We also observed that diffusion and perfusion MRI sequences accurately assess these treatment effects. Indeed, diffusion was a pertinent marker of cell death, and perfusion provided a good indication of the level of necrosis. Additionally, and as expected, T2 MRI was clearly confirmed to be a precise estimator of peritumoral edema.

In studies by Leroy et al. [START_REF] Leroy | Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model[END_REF][START_REF] Tetard | Light Dose Fractionation to Enhance Photodynamic Therapy With 5-Aminolevulinic Acid in Human glioma xenografts[END_REF], the major treatment effect of high-power iPDT was intratumoral necrosis associated with surrounding edema and a higher vascular density corresponding to an inflammatory response in the periphery of the treated tumor. Although the 5-fraction treatment was more effective in terms of tumor-induced necrosis, it was associated with more extensive post-treatment perilesional edema and was thus associated with higher toxicity. Indeed, apoptosis is the preferred cell death pathway for limiting inflammation and excess edema.

In this context, using a light power lower than that used in [START_REF] Leroy | Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model[END_REF][START_REF] Tetard | Light Dose Fractionation to Enhance Photodynamic Therapy With 5-Aminolevulinic Acid in Human glioma xenografts[END_REF] might enhance PDT efficiency by preserving tumor oxygenation [START_REF] Angell-Petersen | Determination of fluence rate and temperature distributions in the rat brain; implications for photodynamic therapy[END_REF][START_REF] Henderson | Fluence rate as a modulator of PDT mechanisms[END_REF]. Additionally, low light power has been suggested to induce selective apoptosis, which causes less damage than is caused by necrosis to normal adjacent brain tissues [START_REF] Alqawi | Molecular mechanisms associated with ALA-PDT of brain tumor cells[END_REF][START_REF] Lilge | Apoptosis induced in vivo by photodynamic therapy in normal brain and intracranial tumour tissue[END_REF][START_REF] Bisland | Metronomic photodynamic therapy as a new paradigm for photodynamic therapy: rationale and preclinical evaluation of technical feasibility for treating malignant brain tumors[END_REF]. Thus, to optimize tumor cytotoxicity while limiting edema and toxicity in adjacent healthy brain tissues, we studied the effects of low-power iPDT on the same murine glioblastoma model. MRI examinations were performed to monitor the low-power iPDT response, and data were combined with previous MRI data acquired alongside the previous high-power iPDT study. This method made a comprehensive analysis of the different treatment schemes feasible.

II. Materials and methods

The main objective of this study was to evaluate the effects of two distinct light delivery regimens delivered at two distinct light powers, leading to four distinct treatment schemes: 1) 2-fractions and 2) 5-fractions delivered at 30 mW each and 3) 2-fractions and 4) 5-fractions delivered at 5 mW each. The same doses of 5-ALA were prescribed, and the same treatment devices and the same light dose of 25 J were used. The resulting four treatment groups were compared to two sham groups: 1) with 5-ALA and without light and 2) without 5-ALA and with light (2 fractions at 5 mW) to assess the effects of either the drug or the laser light alone. -Murine model All specimens were nude rats with a phenotype of Fox n1 rnu/rnu and were obtained from Charles River © (Wilmington, MA, USA) and Envigo® (former Harlan®, Gannat, France).

These nude immune-compromised rats better tolerate experimental xenografts and have no high risk of post-operative infection [START_REF] Chopp | Photodynamic therapy of human glioma (U87) in the nude rat[END_REF]. A quarantine period of 10 days from reception was observed before performing GBM cell grafting. Grafted rats were 8 weeks old and weighed approximately 150 g.

-Grafting

To xenograft the GBM tumors, human GBM cells from the cell line U87 were used. Cell cultures as well as surgical grafting procedures were strictly identical to those described in previous studies [START_REF] Leroy | Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model[END_REF][START_REF] Leroy | MRI assessment of treatment delivery for interstitial photodynamic therapy of high-grade glioma in a preclinical model[END_REF][START_REF] Tetard | Interstitial 5-ALA photodynamic therapy and glioblastoma: Preclinical model development and preliminary results[END_REF]. Surgical grafting was performed under local and general anesthesia administered via a mask delivering isoflurane. A stereotactic frame allowed a micromanipulator to be moved across the 3 planes of space with a 0.01 mm precision. The skull was immobilized horizontally via a tooth bar and two ear bars. A burr hole was drilled at the coordinates mentioned in the Paxinos and Watson atlas (2.70 mm right and 0.50 mm anterior to bregma) to achieve surgical grafting in the right putamen. In all, 5x10 5 U87 glioblastoma cells were injected via a microsyringe connected to an automatic syringe pump.

A cranial anchor was used to guide the needle of the microsyringe and subsequently the optical fiber used for laser light delivery. This anchor was positioned in the burr hole and glued with biological adhesive to the outside of the skull.

b) Treatment

Treatments were delivered 12 days after the xenografts were performed. Animals randomly assigned to one of the treatment groups or to the sham group treated with 5-ALA received an intraperitoneal injection of 5-ALA (100 mg/kg) 4 hours prior to light excitation [START_REF] Olzowy | Photoirradiation therapy of experimental malignant glioma with 5-aminolevulinic acid[END_REF]. Their cages were then covered with a light-occlusive dressing for 24 hours to prevent skin photosensitization (such as erythema or a burn). Then, all animals, regardless of group, were anaesthetized as previously described and placed on the MRI table. A bare flat-end silica fiber UltraSil 272 ULS (OFS, Norcross, USA) with an outside diameter of 350 μm and a numerical aperture of 0.29 was guided into the tumor through the anchor set up during the grafting procedure. T1-weighted MRI was performed to confirm the optimal positioning of the fiber.

Depending on the randomized group, light was delivered interstitially via a 635 nm laser diode. All animals in the treatment groups or the sham group treated with light were subjected to a total dose of 25 J at an irradiance of 30 mW (Figure 1a) or 5 mW (Figure 1b) using variant fractionation schemes that depended on the treatment group (Figure 1). The fractionation regimen used in the 2-fraction group was previously documented by Curnow et al. [START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF]. For this fractionation regimen, the light was stopped after 5 J before the remaining 20 J were delivered. This regimen was reported to produce an area of necrosis more than three times larger than that produced with continuous illumination [START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF]. fractions). The total dose of delivered energy was 25 J at a power of 30 mW or 5 mW (measured at the fiber tip). The following groups are shown: (a) 2 or 5 fractions delivered at a power of 30 mW, (b) 2 or 5 fractions delivered at a power of 5 mW, (c) sham groups treated with 5-ALA/without light to check the hypothetical effect of the drug itself or without 5-ALA/with light to check the hypothetical effect of the laser light itself.

At the end of the procedure, the animals received 3 mg of intraperitoneal methylprednisolone immediately after PDT to limit treatment-induced edema. c) Imaging protocol and analysis A 7 Tesla MRI was used to perform imaging (Bruker, Biospec, Ettlingen, Germany). Seven days after xenografting, tumor growth was assessed using a T2-weighted TurboRARE sequence [START_REF] Schuierer | Fast and ultra-fast MRI of the brain[END_REF] to observe the quality and extent of engraftment. Twelve days after xenografting, the animals received a pretreatment MRI immediately before undergoing iPDT.

Fiber insertion was thus achieved when the tumor graft, as indicated on the MRI, had achieved a minimum coronal diameter greater than 2 mm, and post-treatment MRI was acquired 48-72 hours later.

For follow-up purposes and treatment evaluation, pre-and post-treatment imaging systematically included T2 and T1 perfusion-and diffusion-weighted sequences. T2-weighted TurboRARE sequences [START_REF] Schuierer | Fast and ultra-fast MRI of the brain[END_REF] were acquired in the axial plane to evaluate the geometry and position of the tumor. A T1-weighted sequence was performed before and after gadolinium injection on the axial plane using the RARE type of spin echo. The perfusion sequence was performed using the arterial spin labeling (ASL) [START_REF] Wong | Implementation of quantitative perfusion imaging techniques for functional brain mapping using pulsed arterial spin labeling[END_REF] method. This method provided quantitative information on tissue perfusion by magnetically labeling the blood entering the slice. This corresponded to the wash-in measurement. Diffusion-weighted imaging was achieved in 3D using the Echo-planar Imaging (EPI) technique, which provides information on the degrees of freedom of the water molecules in the tissue.

Post-processing of MRI data for diffusion and perfusion images was performed using Paravision 5.1 software provided by the manufacturer (Bruker©, Biospec, Ettlingen, Germany). Thus, tissue perfusion and diffusion coefficients were measured on axial slices with a thickness of 850 μm and are expressed in units of ml/(100 g tissue/min) and μm 2 /sec, respectively. Then, semiquantitative measurements of diffusion and perfusion values were computed from regions of interest (ROIs) placed 1) on the tumor and 2) contralateral to the tumor (striatum). The ratio between these two ROIs (tumor/healthy brain) was calculated preand post-treatment to calculate the delta (i.e., Δd for diffusion and Δp for perfusion) (Eq. 1).

Eq. 1

x is the coefficient being observed according to the MRI sequence (i.e., diffusion or perfusion) and and , indicates the average coefficient x (i.e., diffusion or perfusion) measured for Tumor_ROI, Striatum_ROI pre-and post-treatment, respectively.

The tumor, necrosis and edema volumes were measured after manual delineation of the T1 and T2 MRI slices (OsiriX 6.5.2, Pixmeo, Switzerland). In particular, tumors were manually delineated via T1 gadolinium MRI, peritumoral edema was delineated on T2 images, and changes in intratumoral signals were manually delineated on either T2 or T1 gadolinium sequences to quantify necrosis.

d) Statistical analysis

Due to the small sample sizes (between 5 and 11 per group), non-parametric tests were used in this study. First, a Kruskal-Wallis test was conducted to evaluate differences in pretreatment tumor volumes among the six groups. If no significant differences were found, a further statistical analysis was performed. This analysis consisted of comparing the delta diffusion, delta perfusion, edema volume and necrosis volume among the six groups using the Kruskal-Wallis test. In cases with significant differences, multiple pairwise comparisons between the corresponding groups were performed using the Dwass-Steel-Critchlow-Fligner post hoc test. Data were analyzed with XLSTAT software 2013 for Windows (Addinsoft, New-York, NY). All significance levels were set to 5%.

III. Results

Forty-eight specimens harbored tumors that were sufficiently large for treatment and underwent post-MRI examination. The median tumor volume, measured from T1 MRI, was 4.00 mm 3 (range, 1.04 to 19.01). The Kruskal-Wallis test showed there were no differences in pre-treatment tumor volumes between the six groups (p=0.503), making further analysis possible.

a) Diffusion

Forty-eight rats benefited from pre-and post-treatment diffusion sequences. All Δd values are represented in Figure 8 and summarized in Table 1. There was no difference in Δd among the six groups (p=0.0549, Kruskal-Wallis test); however, the p-value was slightly higher than 0.05, indicating a tendency toward a difference.

In particular, Δd for the 2-fraction low-power, 5-fraction high-power and 5-fraction lowpower tended to be greater.

b) Perfusion

Among the forty-eight rats, forty-seven benefited from pre-and post-treatment perfusion sequences. All Δp values are represented in Figure 9 and summarized in Table 1. There was a significant difference among the six groups in terms of the Δp (p=0.048, Kruskal-Wallis test), but no pairwise differences were found (due to the small sample sizes, pairwise comparison tests were not statistically powerful and therefore less likely to detect significant differences). However, when data in the 3 groups were pooled (sham, 2-fraction and 5-fraction), a significant difference was found (p=0.0063, Kruskal-Wallis test). The Δp was significantly lower for the 5-fraction group than for the sham group (p=0.002).

c) Necrosis

Among the forty-eight rats, forty-seven benefited from pre-and post-treatment T1 MRI sequences. All necrosis volumes are represented in Figure 10. The Kruskal-Wallis test followed by the Dwass-Steel-Critchlow-Fligner post hoc test revealed that the two sham groups had significantly smaller necrosis volumes than were observed in the four treatment groups (p-values ranging from 0.002 to 0.044). There were no significant differences between the two sham groups or among the four treatment groups (all p-values > 0.40).

d) Peritumoral edema volume

Among the forty-eight rats, forty-seven benefited from pre-and post-treatment T2 MRI sequences. All edema volumes are represented in Figure 11. Using the Kruskal-Wallis test followed by the Dwass-Steel-Critchlow-Fligner post hoc test, we found that the sham groups had significantly smaller edema volumes than were observed in the treatment groups (the two 2-fraction groups treated at 5 mW or 30 mW and the 5fraction group treated at 30 mW) (p-values ranging from 0.002 to 0.05). Moreover, the 5fraction group treated with 30 mW was associated with edema volumes significantly larger than those observed in the 5-fraction group treated with 5 mW (p=0.019). No other significant pairwise differences were found (all p-values higher than 0.05).

IV Discussion

PDT is an emerging cancer treatment that is actively being studied in different body locations.

Optimizing PDT delivery can be undertaken by either increasing 5-ALA uptake and PpIX accumulation [START_REF] Teng | Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy[END_REF] or considering different light regimens that affect tissue oxygenation and may influence free radical production and oxidative species formation [START_REF] Henderson | How does photodynamic therapy work?[END_REF][START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF].

In a previous study [START_REF] Leroy | MRI assessment of treatment delivery for interstitial photodynamic therapy of high-grade glioma in a preclinical model[END_REF], we found that diffusion and perfusion sequences accurately assessed iPDT effects, with diffusion being a relevant marker of cell death and perfusion indicating the level of necrosis. The objective of the current study was to compare previous findings obtained when iPDT was delivered at 30 mW with a new light protocol performed with delivery at a lower power (5 mW). Thus, tumor cell death was evaluated by diffusion imaging, necrosis levels were evaluated by T1 MRI, and perfusion imaging was evaluated for six different treatment schemes. Edema was measured on T2 imaging to grade the toxicity of the treatment delivery strategy.

With regard for diffusion imaging acquired before and after iPDT, Δd was not significantly different among the six groups, but there was a tendency toward a difference (p-value slightly greater than 0.05 (p=0.0549, Kruskal-Wallis test)). In particular, the 2-fraction low, 5-fraction high and 5-fraction low schemes tended to lead to greater Δd. This observation is in line with a previously proposed assumption that the consumption of oxygen influences the photochemical reaction [START_REF] Leroy | Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model[END_REF][START_REF] Leroy | MRI assessment of treatment delivery for interstitial photodynamic therapy of high-grade glioma in a preclinical model[END_REF][START_REF] Tetard | Interstitial 5-ALA photodynamic therapy and glioblastoma: Preclinical model development and preliminary results[END_REF]. Indeed, too much oxygen was probably consumed when light was delivered in only two fractions at a high power. The photochemical reaction was thus limited regardless of the total energy deposited. The importance of fractionation has already been emphasized by Curnow et al. [START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF]., who measured the level of oxygen through an oxygen microelectrode to study the effect of PDT delivered with continuous or fractionated light regimens. They found that the level of tissue oxygen at the treatment site was differentially affected according to light regimens and that this affected the treatment effects.

Those findings are corroborated by our own observations obtained using diffusion imaging, which showed that the best configuration tended to be 5 fractions delivered at 5 mW.

Concerning perfusion imaging, there was a significant difference among the six groups in terms of Δp (p=0.048, Kruskal-Wallis test), but there were no pairwise differences. However, a significant pairwise difference was found (p=0.0063, Kruskal-Wallis test) when the three pooled groups were considered: sham, 2-fraction and 5-fraction (regardless of the light power). Δp for 5 fractions was significantly lower than that observed in the sham group (p=0.002), indicating that 5 fractions led to a more significant effect. These results support the notion that PDT exerts a lesional effect on the tumor vasculature, corroborating the decrease in perfusion values observed within the treated tumors. Additionally, Leroy et al. [START_REF] Leroy | Interstitial photodynamic therapy and glioblastoma: Light fractionation in a preclinical model[END_REF] found that vascular structures were no longer observed within the necrotic areas on histological analysis, corroborating that there was a decrease in perfusion values within the treated tumors when necrosis was measured directly on T1 MRI (Figure 10). In fact, significant pairwise differences were also found in terms of necrosis between the treated and sham groups, but there was no significant difference between the 5-fraction 5 mW group and the 5-fraction 30 mW group. Similarly, in Curnow et al. [START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF], the authors found that fractionation led to up to three times more necrosis than no fractionation when a constant energy deposit was assumed.

The toxicity of the treatment modality (i.e., fractions and light power) was estimated by measuring the volume of edema on T2 MRI. The sham groups had significantly smaller edema volumes than those observed in the two 2-fraction groups and the 5-fraction group treated at 30 mW (p-values from 0.002 to 0.05) but were not significantly different from those observed in the 5-fraction 5 mW group. Additionally, the 5-fraction 5 mW group was associated with significantly lower edema volumes than were found in the 5-fraction 30 mW group (p=0.019), indicating that this treatment modality had a lower toxicity and thus was better tolerated.

According to MRI data and analyses aimed at balancing the treatment effect (level of necrosis by cell deaths characterized by diffusion imaging) and treatment toxicity, the 5-fraction regimen delivered at 5 mW was the most efficient. Indeed, if the 2-fraction treatment at 30 mW is a good candidate for minimizing necrosis, toxicity scored by the edema level was higher than that in the 5-fraction group at 5 mW. Additionally, Δp was lower in the 5-fraction group at 5 mW than that measured in the 5-fraction group at 30 mW, indicating a more pronounced lesional effect.

However, even if MRI allowed a rapid treatment effect analysis, a comprehensive study of the tumor cell death path according to the treatment modality is highly expected to further optimize PDT delivery. Indeed, this preliminary study was conducted in a small number of animals per group and could form a basis for a more complete set of experiments, including oxygen level measurements [START_REF] Curnow | Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes[END_REF], staining for new vessels to explore vascular density (e.g.,

CD31

) and apoptosis staging (e.g., with the TUNEL method, ApopTag® Plus Peroxidase In Situ, Millipore, USA).

Finally, transposing the notion of "high" and "low" energy to humans remains highly challenging. Indeed, the fractionation scheme is easily transposed to humans and has already been proposed in the clinical trial INDYGO [START_REF] Dupont | INtraoperative photoDYnamic Therapy for GliOblastomas: Study Protocol for a Phase I Clinical Trial[END_REF]. However, evaluating the effect of light power is complex, and the choice of optimal power should be carefully balanced. Indeed, the time needed for therapeutic energy deposit may drastically increase the surgery procedure duration. Data collected during the INDYGO trial might help to clarify this question.

V Conclusion

We previously reported that MRI examinations, including diffusion and perfusion sequences, can accurately assess iPDT treatment effects. In the current study, we applied the same MRI imaging protocol to a larger population composed of specimens analyzed in our previous study (i.e., 2-and 5-fractions delivered at 30 mW) and new specimens treated according to different modalities (i.e., 2-and 5-fractions delivered at 5 mW). From observations obtained from MRI data and in consideration of treatment effects (level of necrosis as cell deaths characterized by diffusion imaging) and treatment toxicity, the 5-fraction treatment delivered at 5 mW was the most efficient treatment scheme. In this context, this group might be a good candidate for a more comprehensive exploration of cell death pathways following the delivery of PDT to tumor cells.

  a) Preclinical model -Ethical considerations All operative and animal care procedures were performed in line with the French Government's guidelines (decree 2001-464, 29 May 2001) and those of the Laboratory Animal Care and Use Committee. Our institutional ethics review board approved the study protocol (N° 01878.01).

Figure 1 :

 1 Figure 1: Description of irradiation schemes used in each group (sham, 2 fractions, or 5

Figures 2 , 3 , 4 ,Figure 2 :

 2342 Figures 2, 3, 4, 5 and 6 show illustrations of different groups compared before and after treatment.

Figure 3 :Figure 4 :

 34 Figure 3: Pre-and post-operative MRI examinations of rat #24, which belonged to the sham light-only group. (a) Pre-operative and (b) post-operative T2 MRI; (c) Pre-operative and (d) post-operative diffusion MRI; (e) Pre-operative and (f) post-operative perfusion MRI. Post-operative MRI was performed 72 hours after the introduction of the fiber. (a) and (b): T2, tumor slightly hyperintense without edema. (c) and (d): Slight increase in diffusion within the tumor. (e) and (f): Hyperperfusion of the tumor tissue and cortex.

Figure 5 :Figure 6 :

 56 Figure 5: Pre-and post-operative MRI examinations of rat #32, which belonged to the 2-fraction low-power group. (a) Pre-operative and (b) post-operative T2 MRI; (c) Pre-operative and (d) Post-operative diffusion (e) Pre-operative and (f) Post-operative perfusion MRI. Post-operative MRI was performed 72 hours after the 2-fraction treatment at 5 mW. (b versus a): Large hyposignal area with peritumoral edema. (d versus c): Increase in the diffusion coefficient within the tumor corresponding to necrosis induced by PDT. (f versus e): Increase in perfusion in the peritumoral edema contrasting with a sharp decrease in the central part of the tumor, confirming vascular necrosis induced by PDT.

Figure 7 :

 7 Figure 7: Pre-and post-operative MRI examinations of rat #33, which belonged to the 5-fraction low-power group. (a) Pre-operative and (b) post-operative T2 MRI; (c) Pre-operative and (d) post-operative diffusion MRI; (e) Pre-operative and (f) Post-operative perfusion MRI. Post-operative MRI was performed 72 hours after the introduction of the fiber. (b versus a): Peritumoral edema visible on the T2 image (d versus c): Slight increase in the diffusion coefficient within the tumor corresponding to necrosis induced by PDT. (f versus e): Decrease in perfusion in the center of the tumor, confirming vascular necrosis induced by PDT.

Figure 8 :

 8 Figure 8: Boxplot illustrating Δd for the sham, 2-fraction and 5-fraction groups (red marks represent the median, the size of the box indicates the interquartile range (IQR), and the minimum/maximum are indicated by black marks). The number of specimens per group is shown in brackets.

Figure 9 :

 9 Figure 9: Boxplot illustrating Δp for the sham, 2-fraction and 5-fraction groups (red marks represent the median, the size of the box indicates the IQR, and the minimum/maximum are indicated by black marks). The number of specimens per group is shown in brackets.

Figure 10 :

 10 Figure 10: Boxplot illustrating the volume of necrosis in mm 3 in the sham, 2-fraction and 5-fraction groups (red marks represent the median, the size of the box indicates the IQR, and the minimum/maximum are indicated by black marks). The number of specimens per group is shown in brackets.

Figure 11 :

 11 Figure 11: Boxplot illustrating edema volume in mm 3 in the sham, 2-fraction and 5-fraction groups (red marks represent the median, the size of the box indicates the IQR, and the minimum/maximum are indicated by black marks). The number of specimens per group is shown in brackets.

Table 1

 1 summarizes the specimens included per group, with the measures of each parameter reported for each group.

		Sham	2-Fraction	5-Fraction
		Drug only Light only @30 mW	@5 mW	@30 mW	@5 mW
				(high)	(low)	(high)	(low)
	Specimen	8	9	8	5	8	11
	number
	Δd %	3.41%	2.73%	7.14%	19.70%	18.98%	10.69%
	(IQR)	(1.75, 5.39)	(-0.45, 5.37)	(1.36, 14.88)	(17.59, 23.48)	(11.51, 32.46)	(-5.75, 15.95)
	Δp %	-22.77%	-21.36%	-35.75%	-75.61%	-61.00%	-59.08%
	(IQR)	(-27.17, -	(-31.52, -	(-56.00, -5.81)	(-76.19, -	(-67.99, -	(-84.35, 29.8)
		19.00)	13.77)		12.53)	52.35)
	Necrosis	0	0	2.53	0.77	2.79	1.1
	(mm 3 )	(0, 0)	(0, 0)	(1.00, 5.24)	(0.2, 3.6)	(1.25, 9.24)	(0.1, 3.05)
	Edema	0	0	19.6	21.17	30.6	5.15
	(mm 3 )	(0, 0)	(0, 0)	(13.2, 22.72)	(7.82, 44.90)	(28.62, 40.1)	(0, 7.43)

Table 1 :

 1 Number of specimens eligible for PDT that underwent post-treatment MRI per group (row 2) and summary of Δd and Δp values and necrosis and edema volumes.

https://clinicaltrials.gov/ct2/show/NCT03048240

Financial support provided by the University of Lille, INSERM and the French Society of Neurosurgery.

Ethical approval: This article contains studies performed using animals. All applicable institutional and national guidelines for the care and use of animals were followed. The experimental design was declared to the French National Ethics Committee under reference


Hassiba Belahbib

Zarath M Summers

Marie-Laure Fardeau

Manon Joseph

Christian Tamburini

Alain Dolla

Bernard Ollivier

Fabrice Armougom
email: fabrice.armougom@mio.osuphytheas.fr

Towards a congruent reclassification and nomenclature of the thermophilic species of the genus Pseudothermotoga within the order Thermotogales

Keywords:

The phylum Thermotogae gathers thermophilic, hyperthermophic, mesophilic, and thermo-acidophilic anaerobic bacteria that are mostly originated from geothermally heated environments. The metabolic and phenotypic properties harbored by the Thermotogae species questions the evolutionary events driving the emergence of this early branch of the universal tree of life. Recent reshaping of the Thermotogae taxonomy has led to the description of a new genus, Pseudothermotoga, a sister group of the genus Thermotoga within the order Thermotogales. Comparative genomics of both Pseudothermotoga and Thermotoga spp., including 16S-rRNA-based phylogenetic, pan-genomic analysis as well as signature indel conservation, provided evidence that Thermotoga caldifontis and Thermotoga profunda species should be reclassified within the genus Pseudothermotoga and renamed as Pseudothermotoga caldifontis comb. nov. (type strain = AZM44c09 T ) and Pseudothermotoga profunda comb. nov. (type strain = AZM34c06 T ), respectively. In addition, based upon whole-genome relatedness indices and DNA-DNA Hybridization results, the reclassification of Pseudothermotoga lettingae and Pseudothermotoga subterranea as latter heterotypic synonyms of Pseudothermotoga elfii is proposed. Finally, potential genetic elements resulting from the distinct evolutionary story of the Thermotoga and Pseudothermotoga clades are discussed.

Introduction

Thermotoga maritima [START_REF] Huber | Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90[END_REF] embodies the original member of the phylum Thermotogae, which currently harbors at least 65 cultivated species distributed within 13 genera [START_REF] Jiang | Complete genome sequence and whole-genome phylogeny of Kosmotoga pacifica type strain SLHLJ1T from an East Pacific hydrothermal sediment[END_REF]. Although their true phylogenetic position remains a matter of debate [START_REF] Brochier | Phylogeny: a non-hyperthermophilic ancestor for Bacteria[END_REF][START_REF] Forterre | The universal tree of life: an update[END_REF], the Thermotogales, which are closely related to Aquificales [START_REF] Oshima | Phylogenetic position of Aquificales based on the whole genome sequences of six Aquificales species[END_REF], are commonly depicted as slowly evolving organisms defining an early branching lineage within the Bacteria [START_REF] Achenbach-Richter | Were the original eubacteria thermophiles?[END_REF]. The phylogenetic relatedness among the Thermotogales species do not reflect however the genetic variability of their genomes which undergo pervasive horizontal gene transfer (HGT), including gene influx from Firmicutes and Archaea [START_REF] Nesbø | The genome of Thermosipho africanus TCF52B: lateral genetic connections to the Firmicutes and Archaea[END_REF][START_REF] Nesbø | Evidence for extensive gene flow and Thermotoga subpopulations in subsurface and marine environments[END_REF][START_REF] Zhaxybayeva | On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales[END_REF].

The phylum Thermotogae was originally described from singular characteristics carried by its representatives which thrive most often at high-temperature and exhibit a single-unit lipid membrane as well as a unique loose-fitting sheath structure (called "toga") surrounding the cell [START_REF] Petrus | Genes for the major structural components of Thermotogales species' togas revealed by proteomic and evolutionary analyses of OmpA and OmpB homologs[END_REF]. Until recently, the phylum Thermotogae was believed to mainly comprise anaerobic, thermophilic (T opt ≥ 70 • C) and hyperthermophilic (T opt ≥ 80 • C), fermentative rod-shaped bacteria with the highest growth temperature (65-90 • C) under near-neutral pH conditions [START_REF] Frock | The genus Thermotoga: recent developments[END_REF]. This view was dispelled with the discovery within the order Thermotogales of mesophilic [START_REF] Ben Hania | Cultivation of the first mesophilic representative ("mesotoga") within the order Thermotogales[END_REF][START_REF] Nesbø | Mesotoga prima gen. nov., sp. nov., the first described mesophilic species of the Thermotogales[END_REF] and moderately thermophilic acidophilic lineages comprising the genus Mesotoga and genera Mesoaciditoga and Athalassotoga [START_REF] Itoh | Athalassotoga saccharophila gen. nov., sp. nov., isolated from an acidic terrestrial hot spring, and proposal of Mesoaciditogales ord. nov. and Mesoaciditogaceae fam. nov. in the phylum Thermotogae[END_REF][START_REF] Reysenbach | Mesoaciditoga lauensis gen. nov., sp. nov., a moderately thermoacidophilic member of the order Thermotogales from a deep-sea hydrothermal vent[END_REF], respectively.

The phylum Thermotogae initially consisted within a single class Thermotogae; a single order, Thermotogales; and a single family, Thermotogaceae [START_REF] Reysenbach | Phylum BII. Thermotogae phy. nov. Bergey's Manual of Systematic Bacteriology[END_REF]. Recently however, Bhandari and Gupta have revisited the taxonomy of the Thermotogae by identifying molecular signatures from whole genome-based comparisons [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. The new taxonomic proposal resulted from the identification of Con-served Signature Indels (CSIs) at several taxonomic levels that was fairly congruent with the inference of 16S rRNA-based phylogenetic relatedness of the Thermotogae species [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. According to their findings, the phylum Thermotogae was redefined and divided into four separate orders (Thermotogales, Kosmotogales, Petrotogales and Mesoaciditogales) and five families (Thermotogaceae, Fervidobacteriaceae, Kosmotogaceae, Petrotogaceae and Mesoaciditogaceae). At the genus level, the original genus Thermotoga was proposed to be split into two genera Thermotoga and Pseudothermotoga. This conclusion was supported by 16S rRNA-based phylogenetic reconstruction and identification of clade-specific CSIs that indicated two distinct evolutionary histories of cultivated members of the original genus Thermotoga [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. To date, the genus Pseudothermotoga contains P. elfii, P. lettingae, P. subterranea, P. hypogea and P. thermarum [START_REF] Oren | List of new names and new combinations previously effectively, but not validly, published. List no. 158[END_REF]. Bhandari and Gupta's original work [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF] echoes the modern taxonomy practice that refers to the use of the complete genome DNA sequence as the reference standard to determine both phylogeny and taxonomy of species [START_REF] Rosselló-Móra | Past and future species definitions for Bacteria and Archaea[END_REF]. With the rapid advances in affordable full genome sequencing, the current view of taxonomy thereby integrates whole genome-based comparisons [START_REF] Ramasamy | A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species[END_REF][START_REF] Vandamme | Time to revisit polyphasic taxonomy[END_REF] with an inflow of Overall Genome Relatedness indices (OGRI) [START_REF] Chun | Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea[END_REF] including digital DNA-DNA Hybridization (DDH) [START_REF] Meier-Kolthoff | Taxonomic use of DNA G + C content and DNA-DNA hybridization in the genomic age[END_REF], Average Nucleotide Identity (ANI) [START_REF] Goris | DNA-DNA hybridization values and their relationship to whole-genome sequence similarities[END_REF][START_REF] Konstantinidis | Genomic insights that advance the species definition for prokaryotes[END_REF], or tetra nucleotide regression [START_REF] Teeling | Application of tetranucleotide frequencies for the assignment of genomic fragments[END_REF]. The ANI measure is currently the most accepted OGRI by the scientist community [START_REF] Kim | Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[END_REF].

Recently, two novel thermophilic anaerobic species were isolated from terrestrial hot springs in Japan and were classified as belonging to the genus Thermotoga as T. caldifontis and T. profunda [START_REF] Mori | Thermotoga profunda sp. nov. and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs[END_REF]. Using multiphasic molecular approaches based on 16S rRNA, core-genome phylogeny, and identification of the Pseudothermotoga-specific CSIs, we provide evidence for reclassifying T. profunda and T. caldifontis within the genus Pseudothermotoga as Pseudothermotoga profunda comb. nov., and Pseudothermotoga caldifontis comb. nov., respectively. We also demonstrate by experimental DDH and OGRI measures that Pseudothermotoga lettingae and Pseudothermotoga subterranea should be reclassified as latter heterotypic synonyms of Pseudothermotoga elfii.

Methods

Genome feature analysis

Thermotoga and Pseudothermotoga genomes were retrieved from the NCBI ftp site (ftp://ftp.ncbi.nlm.nih.gov/) under the accession numbers listed in Table 1. The coding sequences (CDS) information was extracted from the gbk and GFF files. The GC content was estimated by the geecee function of the EMBOSS package 6.6.0.0 [START_REF] Rice | EMBOSS: The European Molecular Biology Open Software Suite[END_REF]. The hypothetical protein fractions were deduced from the protein annotation files (faa). The T. caldifontis and T. profunda genomes were included in the genus Pseudothermotoga for the genome feature analysis. Mean comparison of Thermotogaceae clades was performed by the non-parametric Mann-Whitney-Wilcoxon test. The Mann-Whitney-Wilcoxon test can be applied on small sample size and does not require any assumption about the variance equality and population distributions. The Mann-Whitney-Wilcoxon tests, genome size, gene and GC contents were performed by GraphPad prism Software version 5.0. M, SD indicate mean and standard deviation, respectively.

SSU rRNA-based phylogeny

The near-full length 16S rDNA sequences (>1300 pb) of the Thermotogae species were retrieved from the NCBI database (https:// www.ncbi.nlm.nih.gov/) and aligned with the MAFFT software v7.123b [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. A maximum likelihood (ML) phylogenetic tree was constructed from 1624 nucleotide positions using the PhyML 3.1 algorithm [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] implemented in the Seaview package v4.6 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF] with GTR + I + G substitution model and 1000 bootstraps replicates. The phylogenetic tree was rooted using the Coprothermobacter proteolyticus IT3 (GU363592) and Coprothermobacter platensis (Y08935.1) 16S rDNA sequences since the genus Coprothermobacter was the closest branching out group in update SSU rRNA phylogenetic tree [START_REF] Yarza | Update of the all-species living tree project based on 16S and 23S rRNA sequence analyses[END_REF].

Core-genome-based phylogeny

The genome sequences of cultivated Thermotogaceae were retrieved from the NCBI accession numbers summarized in Table 1. A core/pan-genome analysis was performed using the Roary pangenome pipeline v3.6.1 [START_REF] Page | Roary: rapid large-scale prokaryote pan genome analysis[END_REF]. The Roary pipeline generated a core gene alignment from PRANK v.140110 [START_REF] Löytynoja | Phylogeny-aware alignment with PRANK[END_REF] with a 50% identity cut-off level, as well as a gene presence/absence matrix. A maximum-likelyhood (ML) core-genome tree was inferred using PhyML 3.1 algorithm implemented in Seaview v4.6 with generalized time-reversible (GTR) and gamma distribution. PhyML approximated likelihood ratio (aLRT) SH-like values were indicated in a consensus tree. The roary plots.py scripts were used to combine the core-genome-based tree and the gene presence/absence matrix.

Molecular signatures within the genus Pseudothermotoga

The CSIs identified as unique to the genus Pseudothermotoga were originated from reference proteins including the Glycerol-3-phosphate dehydrogenase, the hypothetical protein Theth 0845, the hypothetical protein TM1140 and the hypothetical protein Tlet 0907 [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. The reference protein sequences were retrieved from their corresponding genomes, P. lettingae TMO T (Tlet 907), T. maritima MSB8 T (TM1140), P. thermarum LA3 T (Therth 0845) and P. lettingae TMO T (Glycerol-3-phosphate dehydrogenase). The identification of the homologous proteins in the other Thermotogaceae genomes was performed by BLASTp similarity search [START_REF] Altschul | Basic local alignment search tool[END_REF] using each of the four reference proteins as seed. A protein set was built with each reference protein and their homologs within Thermotogaceae spp. The multiple sequence alignments of the protein sets were performed by MAFFT v.7123b [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. The multiple sequence alignment was visualized by Jalview 2.9.0b2 [START_REF] Waterhouse | Jalview Version 2-A multiple sequence alignment editor and analysis workbench[END_REF] using ClustalX amino acid colors. Only the alignment part containing the CSI region was displayed. The CSI positions were reported according to the reference sequence in each CSI alignment.

DNA-DNA Hybridization dissimilarity measures

DNA-DNA Hybridization (DDH) studies were performed using P. lettingae TMO T (DSM 14385) [START_REF] Balk | Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor[END_REF] and P. elfii SEBR 6459 T (DSM 9442) [START_REF] Ravot | Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oil-producing well[END_REF]. The DDH studies were carried out by the DSMZ as described by De Ley et al. [START_REF] De Ley | The quantitative measurement of DNA hybridization from renaturation rates[END_REF] with some modifications [START_REF] Escara | Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: acceleration of the renaturation rate[END_REF][START_REF] Huss | Studies on the spectrophotometric determination of DNA hybridization from renaturation rates[END_REF] and using a model 2600 spectrophotometer equipped with a 2527-R thermoprogrammer and plotter (Gilford Instruments). Renaturation rates were computed with the TRANSFER.BAS program [START_REF] Jahnke | BASIC computer program for evaluation of GILFORD SYSTEM 2600 spectrophotometer on a PC/XT/AT type personal computer[END_REF].

Whole-genome relatedness indices

The Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) indices were deduced from pairwise genome [START_REF] Konstantinidis | Genomic insights that advance the species definition for prokaryotes[END_REF] and conserved coding proteins [START_REF] Rosselló-Móra | Updating prokaryotic taxonomy[END_REF] comparisons, respectively, using the available full Thermotogaceae genomes. The ANIb measure was preferred to ANIm since it was more appropriate for the comparison of distant related genomes and for Thermotoga spp. [START_REF] Li | The relationship of the whole genome sequence identity to DNA hybridization varies between genera of prokaryotes[END_REF]. The ANIb relied on the sequence alignment tool MUMer [START_REF] Delcher | Fast algorithms for large-scale genome alignment and comparison[END_REF] and was implemented in the JSpecies 1.2.1 software package (www.imedea.uib.es/jspecies). The ANIb range values (95-96%) correspond to the threshold for species delineation [START_REF] Nesbø | The genome of Thermosipho africanus TCF52B: lateral genetic connections to the Firmicutes and Archaea[END_REF]. The AAI calculation was performed by CompareM v0.0.21 software (https:// github.com/dparks1134/CompareM), which uses DIAMOND 0.8.14 [START_REF] Buchfink | Fast and sensitive protein alignment using DIAMOND[END_REF] for best reciprocal hits. Homology was defined with sequence similarity above 50%, sequence coverage above 75% and a minimum E-value of 1e-05. The resulting ANIb and AAI values were transformed in distance matrix and the hierarchical clustering was performed by hclust (R package) using a complete linkage method. The heatmap.2 function (R ggplots package) was used to show the ANI/AAI heatmap with a color scheme relied on ANI values.

Results and discussion

Genomic characteristics within the Thermotogaceae

Previous work revealed that the former genus Thermotoga consisted of two distinct groups of species according to their optimal growth temperature (T opt ) higher than 77 • C and lower than 70 • C for Thermotoga spp. and Pseudothermotoga spp., respectively [START_REF] Frock | The genus Thermotoga: recent developments[END_REF]. With a T opt comprised between 60 • C and 70 • C, Thermotoga caldifontis and Thermotoga profunda shared growth temperature characteristics of Pseudothermotoga spp. In addition to this phenotypic feature, T. caldifontis and T. profunda carried genomic attributes more similar to the Pseudothermotoga than to the Thermotoga representatives (Table 1, Fig. 1). The mean genome size reached 2.12 Mb (SD = 0.073) and 1.84 Mb (SD = 0.044) within the genera Pseudothermotoga and Thermotoga, respectively (Fig. 1A). The genome size of Pseudothermotoga spp. was therefore significantly larger (Mann-Whitney-Wilcoxon test, p = 0.0007) and more variable than the Thermotoga genomes. Moreover, the genome size and growth temperature were negatively associated (r = -0.95) within Thermotogaceae (Table 1). Recently, the "streamlining" concept has been proposed, i.e. that thermal adaptation involves reduction in the genome size [START_REF] Sabath | Growth temperature and genome size in bacteria are negatively correlated, suggesting genomic streamlining during thermal adaptation[END_REF]. As expected, the mean gene content of Pseudothermotoga species compared to Thermotoga species consisted of more coding sequences (Mann-Whitney-Wilcoxon test, p = 0.0007) (Fig. 1B), without affecting genome compactness (similar gene density, data not shown). The hypothetical protein fraction deduced from the CDS functional annotation was larger within the Pseudothermotoga spp. than within the Thermotoga spp. (Table 1). Compared to the GC content of the Pseudothermotoga spp. (M = 42.83, SD = 5.601), those of Thermotoga spp. were slightly higher (not significant, p = 0.3308), and more similar to each other (M = 46.25, SD = 0.463). T. caldifontis and P. hypogea species had the highest GC content (Table 1, Fig. 1C), were closely related but slightly differed from the rest of the Pseudothermotoga spp. because of their lower genome size and a higher GC content.

The phylogenetic relatedness of Thermotogae

The evolutionary relationships of 65 species affiliated to the phylum Thermotogae were established by 16S rRNA-based phylogenetic inference (Fig. 2). The recent description of thermoacidophilic Mesoaciditogales comprising only Mesoaciditoga lauensis and Athalassotoga saccharophila formed the deepest diverging lineage within this phylum [START_REF] Itoh | Athalassotoga saccharophila gen. nov., sp. nov., isolated from an acidic terrestrial hot spring, and proposal of Mesoaciditogales ord. nov. and Mesoaciditogaceae fam. nov. in the phylum Thermotogae[END_REF][START_REF] Reysenbach | Mesoaciditoga lauensis gen. nov., sp. nov., a moderately thermoacidophilic member of the order Thermotogales from a deep-sea hydrothermal vent[END_REF]. The next well-supported branch separated both Petrotogales and Kosmotogales orders from the Thermotogales. The Petrotogales clade consists of Marinitoga spp. as well as the Geotoga and Petrotoga sister groups. Within the order Kosmotogales, the thermophilic Kosmotoga lineage notably harbors the K. olearia and the recent K. pacifica species, which demonstrate an outstandingly wide growth temperature range, 20-79 • C and 33-78 • C, respectively [START_REF] Dipippo | Kosmotoga olearia gen. nov., sp. nov., a thermophilic, anaerobic heterotroph isolated from an oil production fluid[END_REF][START_REF] Jiang | Complete genome sequence and whole-genome phylogeny of Kosmotoga pacifica type strain SLHLJ1T from an East Pacific hydrothermal sediment[END_REF]. The closest relative of Kosmotoga was the mesophilic Mesotoga lineage, with lower growth temperature range (30-40 • C). However, the low bootstrap sup-port (60, Fig. 2) of the Kosmotogales and Petrotogales dichotomy brought into question their true phylogenetic placement [START_REF] Pollo | Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae[END_REF][START_REF] Reysenbach | Mesoaciditoga lauensis gen. nov., sp. nov., a moderately thermoacidophilic member of the order Thermotogales from a deep-sea hydrothermal vent[END_REF] despite a recent core-gene-based phylogeny with 17 Thermotogae species [START_REF] Jiang | Complete genome sequence and whole-genome phylogeny of Kosmotoga pacifica type strain SLHLJ1T from an East Pacific hydrothermal sediment[END_REF]. Finally, Thermotogales appeared to be the most diverse lineage, encompassing the genera Thermotoga, Pseudothermotoga, Thermosipho, Oceanotoga and Fervidobacterium. Within the order Thermotogales, the Thermotoga and Pseudothermotoga clades shared an exclusive common ancestor (red circle, Fig. 2); they were sister groups and were therefore more closely related to one another than to Thermosipho spp. From their most recent ancestor, more evolutionary change was observed within the Pseudothermotoga than within the Thermotoga lineage. However, the weakness of the phylogenetic signal from the 16S rRNA-based phylogeny did not offer an acceptable means to determine the relationships between Thermotoga species (Fig. 2). The poorly resolved phylogeny of the Thermotoga representatives indicates a need to investigate additional phylogenetic markers (RpoB), core-genes phylogeny [START_REF] Jiang | Complete genome sequence and whole-genome phylogeny of Kosmotoga pacifica type strain SLHLJ1T from an East Pacific hydrothermal sediment[END_REF] or concatenated housekeeping proteins [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. Additionally, a full comparative genomics study of the gene flow events undergone by the members of the two genera would shed light on their evolutionary history. This first phylogenetic approach suggested that Thermotoga profunda and Thermotoga caldifontis belong to the Pseudothermotoga clade rather than to Thermotoga (Fig. 2).

The core-genome-based phylogeny of Thermotogaceae

To confirm the taxonomic affiliation of Thermotoga caldifontis and Thermotoga profunda within the genus Pseudothermotoga, we investigated large-scale features of their genomes. The inference of core-genome-based phylogeny of the family Thermotogaceae relied on the analysis of the gene content of eight Thermotoga and six Pseudothermotoga species. The identification of the genes shared by all the available Thermotogaceae genomes (the core-genome) was deduced by gene orthology and resulted in 614 genes used for the Thermotogaceae phylogeny reconstruction. The core-genomebased phylogeny preserved P. hypogea as the closest relative of T. caldifontis, and the sister taxa P. elfii as the closest relatives of T. profunda (Fig. 3), as was observed in the 16S rRNA-based phylogeny (Fig. 2). The lack of consistency in the tree topology of Thermotoga spp. between 16S-rRNA-based and core-genome-based phylogeny can be explained by the low bootstrap branch supports for this clade (Fig. 2). In Fig. 3, primary genetic indicators of the dichotomy of genera Pseudothermotoga and Thermotoga were denoted with a gene presence (blue tile)/absence matrix, where the genes shared by two or more species are shown by aligned blue tiles or blocks. Similar gene content profiles of Thermotoga species resulted in a considerable core-genome size (number of genes shared by all the members) to the detriment of the accessory genome (shared by at least two members but not by all) and species-specific genes (Fig. 3). In contrast, the Pseudothermotoga species showed more variability in their gene content profiles, which was exhibited by a lower core genome size but a greater accessory genome and more species-specific genes (Fig. 3). The gene content showed that T. profunda and T. caldifontis were genetically more closely related to the Pseudothermotoga than to Thermotoga species. As discussed above, deciphering the distinct evolutionary history of the Thermotoga and Pseudothermotoga lineages will require more complex compara-

5.

Whole-genome relatedness within the Thermotogaceae family. Hierarchical clustering from Average Nucleotide Identity (vertically) and Average Amino Acid Identity (horizontally) measures using 19 Thermotogaceae genomes. Value scale is depicted in red, yellow and green colors for ANI genome pairwise comparison. The value scale ranges from 0 to 100% according to sequence identity level. The vertical line at the value 96 indicates the ANI species demarcation threshold. tive genomics including gene family reconstruction, identification of species-specific genes, functional systems, and horizontal gene transfer, a dominant force of Thermotogales evolution [START_REF] Nesbø | Evidence for extensive gene flow and Thermotoga subpopulations in subsurface and marine environments[END_REF].

Molecular signatures of the Pseudothermotoga clade

Previous genomic insights on the Thermotogae have brought out the discovery of four Conserved Signature Indels (CSIs) specific to the members of the Pseudothermotoga clade [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. The Pseudothermotoga-specific CSIs have been identified from Thermotogales protein family alignments of the Glycerol-3-phosphate dehydrogenase (one amino-acid insertion), the hypothetical Theth 0845 (5/6 amino-acid insertion), the hypothetical TM1140 (one amino-acid deletion) and the hypothetical Tlet 0907 (five amino-acid deletion) [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. Presence of the four Pseudothermotogaspecific CSIs within Thermotoga profunda and Thermotoga caldifontis offers additional evidence of their reclassification proposal as Pseudothermotoga species. The multiple sequence alignment (MSA) of the homologs to the Glycerol-3-phosphate dehydrogenase (Fig. 4A) and to the hypothetical protein TM1140 (Fig. 4D) showed that the two Pseudothermotoga-specific CSIs, a Leucine insertion and a Leucine deletion, respectively, were also conserved in T. caldifontis and T. profunda (Fig. 4A,D). The next Pseudothermotoga-specific CSI, a five-amino-acid deletion derived from the MSA of homologs to the Tlet 0907 protein, was also shared by both T. caldifontis and T. profunda (Fig. 4B). However, Bhandari and Gupta reported a six-amino-acid deletion in their MSA of homologs to Tlet 907 [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF]. In the same way, the MSA of homologs to the Theth 0845 protein exhibited also a slight modified pattern from the original Pseudothermotoga-specific CSI described in supplementary Fig.

of Bhandari and Gupta work [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF] (Fig. 4C).

Our analysis led to identify two slight modified Pseudothermotoga-specific CSIs 4B,C) compared to the original work that could be explained by the MSA methods used and by the divergence of the selected sequences in the MSA. Using the T-COFFEE method [START_REF] Armougom | Expresso: automatic incorporation of structural information in multiple sequence alignments using 3D-Coffee[END_REF] (data not shown), the CSIs identified were congruent with our MAFFT-based analysis. The accuracy evaluation of the MSA (benchmark) commonly shows that consistency-based programs such as T-COFFEE and MAFFT exceed the other methods [START_REF] Pais | Assessing the efficiency of multiple sequence alignment programs[END_REF]. It is also important to emphasize that the previous identification of Pseudothermotoga-specific CSIs [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF] involved only two Pseudothermotoga spp. (P. lettingae and P. thermarum) whereas all Pseudothermotoga spp. were included in our analysis (Fig. 4). Finally, the pairwise identity matrix of the Theth 0845 protein family alignment (Fig. 4C) provided low sequence identity values (<35%) close to the twilight zone [START_REF] Rost | Twilight zone of protein sequence alignments[END_REF] that could stimulate debate about the true evolutionary relatedness of these proteins and especially the biological relevance of this CSI. The occurrence of the four Pseudothermotoga-specific CSIs in T. caldifontis and T. profunda is thereby an additional argument to reclassify these two organisms within the genus Pseudothermotoga as P. caldifontis comb. nov. and P. profunda comb. nov., respectively.

Species delineation within the genus Pseudothermotoga Fardeau et al. [START_REF] Fardeau | Archaeoglobus fulgidus and Thermotoga elfii, Thermophilic isolates from deep geothermal water of the Paris Basin[END_REF], previously reported that DNA-DNA homologies between P. elfii strain G1 and P. subterranea DSM 9912 T , P. lettingae DSM 14385 T and P. elfii DSM 9442 T were 83%, 71.4% and 86%, respectively. This study also indicated a DNA-DNA homology of 83% between P. subterranea DSM 9912 T and P. elfii DSM 9442 T , and of 78% between P. subterranea DSM 9912 T and P. lettingae DSM 14385 T [START_REF] Fardeau | Archaeoglobus fulgidus and Thermotoga elfii, Thermophilic isolates from deep geothermal water of the Paris Basin[END_REF]. In our study, complementary DDH experiments were carried out in duplicate with P. lettingae DSM 14385 T and P. elfii DSM 9442 T , they indicated DNA-DNA homology of 94% (91.5% and 96.7%) between these two species. All DDH values of the pairwise species comparison were thereby above 70%. Consequently, all the Pseudothermotoga strains could not be differentiated at the species level by DDH when taking into account the recommendation of the ad hoc committee [START_REF] Wayne | Report of the ad hoc committee on reconciliation of approaches to bacterial systematics[END_REF], which advised a threshold value of 70% DDH similarity for the definition of bacterial species.

The close relationship linking DDH and ANI values [START_REF] Goris | DNA-DNA hybridization values and their relationship to whole-genome sequence similarities[END_REF][START_REF] Kim | Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[END_REF] led us to calculate ANI indices for estimating overall genome similarity and clarifying the genetic relationships within the genus Pseudothermotoga. In addition to ANI indices, the Amino Acid Identity (AAI) measure was also considered for its robustness for divergent species [START_REF] Rosselló-Móra | Updating prokaryotic taxonomy[END_REF]. Within the Thermotogaceae genomes, the estimated ANI and AAI values ranged from 64.19 to 99.96% and from 63.32 to 99.98%, respectively. The double ANI-AAI clustering distinguished the Pseudothermotoga and Thermotoga genera (Fig. 5). The Thermotoga clustering showed intra-species relationships for Thermotoga sp. Xyl 54, Thermotoga. sp Cell2, Thermotoga. sp. TBGT1765 and Thermotoga. sp TBGT1766 (AAI and ANI values >98). Strikingly, both ANI and AAI values revealed intra-species relationships between P. elfii and P. lettingae with 99.12% and 99.5% of identity level, respectively. Based upon DDH and ANI-AAI results, P. elfii, P. lettingae and P. subterranea species should be considered as heterotypic synonyms. Herein we propose to unite these three species. According to rules 38, 42 and 24b(2) of the Bacteriological Code [START_REF] Parker | International code of nomenclature of prokaryotes[END_REF], the name Pseudothermotoga elfii has priority and hence should be used for the unified taxon.

Conclusions

With the significant advances in full genome sequencing, modern taxonomy practice promotes the use of gene markerbased/core-genome-based phylogenetic inferences, comparative genomics, and whole genome relatedness indices as reliable methods for taxonomic classification [START_REF] Chun | Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea[END_REF][START_REF] Ramasamy | A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species[END_REF][START_REF] Schleifer | Taxonomy in the age of genomics[END_REF][START_REF] Vandamme | Time to revisit polyphasic taxonomy[END_REF]. As a result, investigation of the Thermotoga and Pseudothermotoga genera by modern taxonomy practice and classical DDH measures puts forward congruent classification and nomenclature of these genera within the Thermotogaceae family. Our data show that Thermotoga caldifontis and Thermotoga profunda species share similar features (genome size, CDS number) with the Pseudotermotoga species. They are also phylogenetically related to Pseudothermotoga species, and carry the four Pseudothermotoga-specific CSIs. Moreover, a preliminary pan-genomic analysis shows that T. profunda and T. caldifontis are genetically more closely related to the Pseudothermotoga spp. We thereby propose to reclassify Thermotoga caldifontis and Thermotoga profunda as Pseudothermotoga caldifontis comb. nov. and Pseudothermotoga profunda comb. nov., respectively. We also demonstrate that Pseudothermotoga elfii, P. subterranea and P. lettingae are heterotypic synonyms and should therefore be united with the priority name of Pseudothermotoga elfii. Despite the considerable effort needed to understand the Thermotogae phylogenetic relationships [START_REF] Forterre | The universal tree of life: an update[END_REF][START_REF] Zhaxybayeva | On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales[END_REF], the evolutionary events driving Thermotogae sub-groups remain blurred. Further complex comparative genomics may assist the deciphering of the evolutionary mechanisms within Thermotogae. The description of this taxon is the same as that given previously [START_REF] Mori | Thermotoga profunda sp. nov. and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs[END_REF].

Description of

The type strain is AZM34c06 T = NBRC 106115 T = DSM 23275 T .

The sequence accession number (16S rRNA gene) for the type strain is NR 133904.1.

The accession number of the genome sequence is AP014510. The description of this taxon is the same as that given previously [START_REF] Mori | Thermotoga profunda sp. nov. and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs[END_REF].

Description of

The type strain is AZM44c09 T = NBRC 106116 T = DSM 23272 T .

The sequence accession number (16S rRNA gene) for the type strain is NR 133903.1.

The accession number of the genome sequence is AP014509.

Emended description of the genus Pseudothermotoga Bhandari and Gupta 2014

The morphological and physiological description of this genus, whose type species is Pseudothermotoga thermarum [START_REF] Bhandari | Molecular signatures for the phylum (class) Thermotogae and a proposal for its division into three orders (Thermotogales, Kosmotogales ord. nov. and Petrotogales ord. nov.) containing four families (Thermotogaceae, Fervidobacteriaceae fam. nov., Kosmotogaceae fam. nov. and Petrotogaceae fam. nov.) and a new genus Pseudothermotoga gen. nov. with five new combinations[END_REF], remains the same as described by Windberger et al. [START_REF] Windberger | Thermotoga thermarum sp. nov. and Thermotoga neapolitana occurring in African continental solfataric springs[END_REF]. Other species belonging this genus are P. profunda (Basonym: T. profunda) and P. caldifontis (Basonym: Thermotoga caldifontis) (Mori et al. [START_REF] Mori | Thermotoga profunda sp. nov. and Thermotoga caldifontis sp. nov., anaerobic thermophilic bacteria isolated from terrestrial hot springs[END_REF]).

Emended description of the species Pseudothermotoga elfii Bhandari and Gupta 2014

The characteristics of this species are as described by Ravot et al. [START_REF] Ravot | Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oil-producing well[END_REF] with the following amendments. The following substrates may be used: methanol, lactate, pyruvate, galactose, cellobiose, glycerol, starch, xylan, pectin, methylamine, dimethylamine, trimethylamine, 2-oxoglutarate, serine, yeast extract, peptone, gelatin and casamino acids. Acetate, betaine, leucine, isoleucine, valine, formate and H 2 /CO 2 (80/20, v/v) may be oxidized either in the presence of thiosulfate or a hydrogenotrophic methanogen as electron acceptor. Elemental sulfur, Fe(III) and anthraquinone-2,6disulfonate may serve as electron acceptors. The type strain is DSM 9442 T = ATCC 51869T.

Fig. 1 .

 1 Fig. 1. Genome features within Thermotogaceae family. (A) Genome size variability. (B) CDS number distribution. (C) GC content distribution. Black line indicates mean ± SD. T. caldifontis and T. profunda species are included within the genus Pseudothermotoga.

Fig. 2 .

 2 Fig. 2. 16S rRNA-based phylogenetic tree of the Thermotogae. ML tree built from 65 16S rRNA sequences and 1624 positions. Bootstrap values from 1000 replicates are indicated only when >50%. The tree is rooted with 16S of Coprothermobacter proteolyticus IT3 and Coprothermobacter platensis. Bar scale, 9% nucleotide substitutions. Order and family taxonomic levels are indicated in black and blue, respectively. The Pseudothermotoga and Thermotoga genera are boxed in red; the red circle indicates their exclusive common ancestor. Details of the branch support values of phylogenetic relatedness within Pseudothermotoga and Thermotoga is shown by a cladogram on the top right.

Fig. 3 .

 3 Fig. 3. Core-genome-based phylogeny of the Thermotogaceae family. The core-genes consensus midpoint tree (left) is deduced from 603,752 positions of 614 shared genes. PhyML branch support values are indicated in black respectively. The gene presence/absence matrix is deduced from 5052 total genes (right). Blue tile shows the presence of gene in a genome. Aligned blue tiles are genes shared by a least two genomes. Pseudothermotoga genus is boxed in red. The pan-genomic profile for the Pseudothermotoga and Thermotoga clades are shown as bar charts.

Fig. 4 .

 4 Fig. 4. Partial sequence alignments of the Pseudothermotoga-specific Conserved Signature Indels. Partial alignments extracted from full protein sequence alignments. The positions added to sequence labels mean start/end positions according to the full aligned sequences. Positions on the top of the CSIs boxed in dashed red lines target the reference sequence (labeled in bold). T. caldifontis and T. profunda hold the four Pseudothermotoga-specific CSIs. (A) Glycerol-3-phosphate dehydrogenase. A Leucine insertion conserved in all Pseudothermotoga members. (B) Hypothetical protein Tlet 0907. Pseudothermotoga members show a specific five-amino acid deletion. (C) Hypothetical protein Theth 0845. A five-amino-acid insertion is conserved within the Pseudothermotoga clade. (D) Hypothetical protein TM1140. A Leucine deletion conserved within the Pseudothermotoga clade.

  Pseudothermotoga profunda comb. nov. Pseudothermotoga profunda (pro.fun'da L. fem. adj. profunda deep, pertaining to the source of isolation of the type strain) Basonym: Thermotoga profunda Mori et al. 2014.

  Pseudothermotoga caldifontis comb. nov. Pseudothermotoga caldifontis (cal.di.fon'tis. L. adj. caldus hot; L. masc. n. fons, fontis a spring, N.L. gen. n. caldifontis of a hot spring, pertaining to the source of isolation of the type strain) Basonym: Thermotoga caldifontis Mori et al. 2014.

Table 1

 1 Genomic and growth features within the Thermotogaceae.

	Strains	Genome size (bp)	GC content (%)	Predicted CDS	CDS without predicted	Optimal growth	Accession number
					function	temperature
	P. elfii DSM 9442 T	2,169,860	39	2063	497 (24%)	66	AP014507
	P. lettingae TMO T DSM 14385	2,135,342	39	2047	414 (20%)	65	CP000812
	T. profunda AZM34c06 T	2,187,612	39	2073	600 (29%)	60	AP014510
	P. hypogea DSM 11164 T	2,165,416	49	2073	663 (32%)	70	CP007141
	T. caldifontis AZM44c09 T	2,014,912	51	1940	564 (29%)	70	AP014509
	P. thermarum DSM 5069 T	2,039,943	40	1965	428 (21%)	70	CP002351
	T. naphthophila RKU-10 T	1,809,823	46	1805	333 (19%)	80	CP001839
	T. petrophila KU-1 T	1,823,511	46	1772	335 (19%)	80	CP000702
	Thermotoga sp. Cell2	1,749,904	46	1632	321 (18%)	-	CP003409
	Thermotoga sp. RQ2	1,877,693	46	1832	337 (18%)	80	CP000969
	Thermotoga sp. RQ7	1,851,618	47	1812	347 (19%)	80	CP007633
	T. neapolitana DSM4359 T	1,884,562	47	1829	354 (19%)	80	CP000916
	Thermotoga sp. 2812B	1,843,731	46.2	1808	361 (20%)	-	CP003408
	T. maritima MSB8 T	1,869,612	46	1858	369 (20%)	80	AE000512

Acknowledgement

We thank the Professor William B. Whitman, University of Georgia, for the revision of the manuscript.

Funding

This project has been funded with support from ExxonMobil Research and Engineering.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.syapm.2018.04. 007.


Minouche Knoepffler-Péguy

Charles-François Boudouresque

Michèle Knoepffler

Suzanne Berthier

-2018), the 'Dame de Banyuls' (the Lady of Banyuls)

This painting shows her at the age of 19, in a ball dress, in Tunis (Tunisia). Portrait painted by Geneviève Gavrel .

Minouche wanted to do a doctorate in zoology; but when she went to see the professor who might have given her a project in this field, she went the wrong door: she knocked at the office of Jean Feldmann. Jean Feldmann was at the time one of the best known phycologists in Europe. His charisma was such that she left his office with a doctoral research subject focusing on the Ectocarpaceae, a 'diabolical' group of brown algae (Phaeophyceae), today placed within the kingdom of the Stramenopiles [START_REF] Boudouresque | Taxonomy and phylogeny of unicellular eukaryotes[END_REF]. The Ectocarpaceae constitute a 'diabolical' taxon because of the unbelievable complexity of their biological cycles, where almost ten generations may succeed each other, their morphological plasticity according to environmental factors and the rarity of characters that allow proper diagnosis between the species; at the time of Minouche's doctoral thesis, genetic tools did not yet exist; today, these tools are used, but it is not certain that they have succeeded in 'de-diabolising' the taxonomy of the Ectocarpaceae. Minouche's doctoral research was carried out at Banyuls-sur-Mer (French Catalonia). Her thesis entitled 'Recherches sur le polymorphisme en culture de quelques Ectocarpacées (Feldmannia et Acinetospora)' was defended in 1974 in Paris, before an examining committee consisting of Jean Feldmann, Marius Chadefaud, Francis Magne, René Nozeran and Pierre Drach; she was awarded her doctorate with the distinction 'très honorable avec félicitations du jury', the highest distinction awarded by French universities.

Minouche's research was at first focused on the Ectocarpaceae (the subject of her doctoral thesis). Subsequently, she also took an interest in the Cystoseira. The Banyuls-sur-Mer region, at that time, was a hotspot of diversity for this genus of brown algae [START_REF] Feldmann | Recherches sur la végétation marine de la Méditerranée : la côte des Albères[END_REF]; since then, many species of Cystoseira have been considered as locally extinct in French Catalonia [START_REF] Pinedo | Long-term decline of the populations of Fucales (Cystoseira spp. and Sargassum spp.) in the Albères coast (France, North-western Mediterranean)[END_REF] [START_REF] Boudouresque | L'algue japonaise Undaria pinnatifida (Phaeophyceae, Laminariales) en Méditerranée[END_REF]69 citations), [START_REF] Boudouresque | Inventaire des algues marines benthiques dans les Pyrénées-Orientales (Méditerranée, France)[END_REF]41 citations) and [START_REF] Knoepffler-Péguy M | Sargassum muticum begins to invade the Mediterranean[END_REF]41 citations).

Minouche loved teaching. While very young, she taught as an assistant for laboratory practical work in Tunis (Tunisia), then in Paris (France); she was also a peripatetic teacher (maîtresse auxiliaire) in French secondary schools, replacing teachers who were on sick leave or maternity leave. The rest of her university career [Assistante (Junior Lecturer), then Maître-Assistante (Lecturer) and finally Maître de Conférences (Senior Lecturer)] has already been outlined. The course in marine phycology and ecology (hereafter 'phycology course') at Banyuls-sur-Mer constituted the great adventure of her professional life. This course was set up in 1950 by Prof. Jean Feldmann; René Delépine was the organiser, and Minouche was associated with it from 1968. In 1972, when René Delépine was transferred (at his request) to Villefranche-sur-Mer (another of the Université Pierre et Marie Curie marine sciences research stations), Minouche took the lead of the phycology course. This course played a major role in the development of Mediterranean phycology. At first, it was opened to students from the Université Pierre et Marie Curie (Paris); but it soon took on an international dimension; it began by accepting students from other French universities, then from other universities in European and Mediterranean countries; finally, it became officially a European course (with the European Union label Erasmus, then Socrates) (Fig. 2,3). The teaching staff, in particular Francesco Cinelli (Italy), Andrea Cossu (Sardinia), Hans Frei (Germany), Vittorio Gazale (Sardinia), Amelia Gómez, María Antonia Ribera, Conxi Rodríguez-Prieto (Catalonia), Marie-Claude Noailles (Paris) and myself, came from all over Europe. The Banyuls course was attended by hundreds of students, young researchers and teachers; it would be hard to find a country in Europe or around the Mediterranean where there was not at least one person who had attended the Banyuls-sur-Mer course. Overall, in Western Europe and the Mediterranean basin, large numbers of people who are now researchers, teachers or managers have taken this course; they did not all become phycologists, however they all have rich memories of the course. Minouche made this course into not only a learning experience in phycology and marine ecology, but also an experience that was fun, inclusive and a hotspot of cultural exchange. The highpoint of the course, which was held over three weeks in July and August, was the 'international meal'; in her large house in the Rue Barra, she and her husband and children welcomed not only the lecturers and students from the course, but also the researchers from the Laboratoire Arago. Each group of course students prepared a dish from their country: the Spanish paella stood beside the Corsican lonzu, the Moroccan tajines, the Greek moussaka and the Venitian linguini alle vongole. Minouche presided like a queen in the centre of the apartment, surrounded by students and colleagues; she was happy when she saw people being happy. Minouche's successors, after her retirement in 2001, were not able or willing to carry on with the Banyuls-sur-Mer phycology course; perhaps they possessed neither her general competence in taxonomy, nor her charisma that enabled her to mobilise the Mediterranean phycologists. And perhaps this course was too much of an overlap with the one held at Roscoff (another marine sciences research station run by the Université Pierre et Marie Curie). Perhaps finally, in the age of the all-powerful DNA sequencing (a precious tool, powerful and of proven value), this course had lost its raison d'être. Paradoxically, as Boero noted (2016aBoero noted ( , 2016b)), the famous Rio summit in 1992, and the Convention on Biological Diversity, have not led to a surge in taxonomy, but rather to its decline, in terms of posts and budgets. The result is a disaster (for the sciences of the environment) [START_REF] Hutchings | An advocate for taxonomic research in Australia[END_REF]. But there is always room for a swing in the other direction, a return of the pendulum; now it is coming from the USA, where the disaster has been fully perceived (Boero, 2016a(Boero, , 2016b;;[START_REF] Blanfuné | Une idée neuve, la taxonomie (Nomina si nescis, perit cognitio rerum)[END_REF].

On the occasion of the 14 th International Seaweed Symposium, at Brest and Saint-Malo (France), Minouche organised (with Marie-Claude Noailles and the author of the present obituary) the pre-symposium excursion to the Western Mediterranean, at Banyuls-sur-Mer, from 10th to 15th August 1992.

In 1967, Michèle Peguy married Louis-Philippe Knoepffler (1926[START_REF] Boudouresque | Inventaire des algues marines benthiques dans les Pyrénées-Orientales (Méditerranée, France)[END_REF], a herpetologist of international repute, Directeur de Recherches at the CNRS (Centre National de la Recherche Scientifique), a major French scientific research organisation. They had five children: Frédérique (nicknamed 'Frika';[START_REF] Péguy | Sur la croissance en culture de quelques individus de Cystoseira mediterranea (Ag.) Sauv. en vue de l'étude du cycle de reproduction d'une Ectocarpacée épiphyte[END_REF]), Ragnar (1967)), Harald (1971), Herrade (1974) and Sieghilde (1976). Minouche died on Friday 14 September 2018, at the age of 84.

For her collaborators who were with her on the Banyuls course, Minouche was sometimes difficult to live with. She was to all appearances the soul of disorder; she made everything complicated, to the point that we nicknamed her 'Why make things simple when you can make them complicated?'. Her office accommodated a mountain of documents, from floor to ceiling, where you could hardly move and where a cat would have had trouble finding her kittens; and she could never find anything. But thanks to her charisma, she always got what she wanted: in Brussels and in Strasbourg (the European capitals), in Paris and at Banyuls, many people detested the confusion of her projects, her apparent or real disorganisation; and yet, her projects were accepted in the end. Minouche did not speak English, or if she did it was a kind of unbelievable pidgin English, a mixture of French words pronounced in a more or less English way and English words pronounced in a French way; and yet, many of her audience had a good laugh but understood her. I admit that I, who put some effort into speaking a rather more academic form of English, and despite that I am often misunderstood, I was a little vexed and jealous of her because of this.

Minouche was a practising Catholic, who would not miss the Sunday mass for anything in the world; she professed in particular a deep devotion to Mary, mother of Jesus. At the same time, Minouche was extraordinarily tolerant and respectful of other cults and religions and of nonbelievers. The way she managed the phycology course, a real melting pot of all the Mediterranean cultures, testifies to this. Minouche also loved cats; they ruled as true masters in her house; in French, besides, 'minouche' is a name often given to female cats. Finally, she had a special relationship with frogs, which she collected representations of, following in the footsteps of her husband Louis-Philippe. According to her daughter Frika, she owned more than 700 of them. Michèle Knoepffler-Péguy was certainly not among the most prestigious of 20th century phycologists and ecologists. This was in any case never her ambition. She described few species, she did not revolutionise our vision of the world of algae and she was not a precursor of the ecosystem-based approach [START_REF] Personnic | An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow[END_REF][START_REF] Boudouresque | L'approche écosystémique. Un petit pas pour l'homme, un grand pas pour la gestion de la nature[END_REF][START_REF] Thibaut | An ecosystem-based approach to assess the status of Mediterranean algaedominated shallow rocky reefs[END_REF]. Her legacy resides rather in the Banyuls phycology course, which laid new foundations in Europe, and which will perhaps prove to be the foundation of a renewal of taxonomy (Boero, 2016b;[START_REF] Boudouresque | L'approche écosystémique. Un petit pas pour l'homme, un grand pas pour la gestion de la nature[END_REF][START_REF] Hutchings | An advocate for taxonomic research in Australia[END_REF][START_REF] Blanfuné | Une idée neuve, la taxonomie (Nomina si nescis, perit cognitio rerum)[END_REF]. This would indeed be a fine homage to Minouche.

Figure 1 .

 1 Figure 1. Michèle Péguy ('Minouche'). This painting shows her at the age of 19, in a ball dress, in Tunis (Tunisia). Portrait painted by Geneviève Gavrel.

Figure 2 .

 2 Figure 2. Michèle Knoepffler-Péguy ('Minouche') (right), in the practicals lab at the Laboratoire Arago, with phycology course students, Delphine Willsie (left) and X (back view), July 1981. Photo © Boudouresque.

Figure 3 .

 3 Figure 3. Michèle Knoepffler-Péguy ('Minouche'), giving a class to students on the phycology course, in the practicals lab at the Laboratoire Arago, July 1990. Photo © Charles-François Boudouresque.

  . Finally, Minouche worked on biological invasions (Sargassum muticum and Undaria pinnatifida). She described one species: Pedobesia solieri Feldmann ex Abélard & Knoepffler. She also proposed two new combinations: Feldmannia caespitula (J. Ag.) Knoepffler-Péguy (now: Feldmannia paradoxa (Montagne) Hamel var. donatiae (Ercegović) Antolić & Span) and Feldmannia caespitula var. lebelii (Areschoug ex P. Crouan & H. Crouan) Knoepffler-Péguy (now: Feldmannia lebelii (Areschoug ex P. Crouan & H. Crouan) Hamel). The four most often cited of Minouche's publications are: Boudouresque et al. (1992; 77 citations),

Acknowledgements. Michèle Perret-Boudouresque, curator of the Plateforme Macrophytes at the Mediterranean Institute of Oceanography (MIO; Aix-Marseille University) helped me to reconstitute the list of works by Michèle Knoeepffler-Péguy. Frédérique Knoepffler ('Frika', her daughter), designated as my contact within the family by her siblings (Ragnar,

Minouche spent her childhood in Tunis, she studied there and get her Baccalauréat (year undergraduate course, in France and in Tunisia during the French colonial period; 1952)Paris (Université Pierre et Marie Curie), and was awarded the Licence-ès-Sciences