The genus Peptoniphilus was created by Ezaki et al. (Ezaki et al., 2001) in 2001 by the transfer of five Peptostreptococcus species into this new genus based on their branching in the 16S rRNA gene tree. The genus was subsequently placed into the family Peptoniphilaceae within the order Tissierellales (Johnson et al., 2014). Peptoniphilus species comprise Gram-positive anaerobic cocci (GPAC), which are common constituents of various human polymicrobial infections (Wan et al., 2021, Brown et al., 2014, Lu et al., 2022, Willner et al., 2014). These species are generally commensals of human gut and vagina, as well as skin, mouth and upper respiratory tract, with most having been isolated from human clinical samples (Ezaki et al., 2001, Wan et al., 2021, Diop et al., 2019, Mishra et al., 2012, Cho et al., 2015). Peptoniphilus spp. either alone or as parts of polymicrobial infection are also involved in blood stream infections (Wan et al., 2021, Brown et al., 2014, Lu et al., 2022), and a recent study indicates that the presence of these spp. in urine and prostate microbiomes shows association with increased risk to prostate cancer (Hurst et al., 2022).

The genus Peptoniphilus originally contained only five species including the type species P. asaccharolyticus, as well as P. indolicus, P. lacrimalis, P. harei, and P. ivorii (Ezaki et al., 2001). However, since then large numbers of new species have been identified and added to this genus (Wan et al., 2021, Diop et al., 2019, Mishra et al., 2012, Cho et al., 2015, Aujoulat et al., 2021;44(5):126235., Ryu et al., 2021, Beye et al., 2018, Patel et al., 2016). According to the LPSN (Parte et al., 2020), the genus Peptoniphilus presently comprises 33 species, of which 23 species names (including two synonyms) are validly published, and the remaining 10 species viz. “P. coli” (Mbaye et al., 2022), “P. mikwangii” (Cho et al., 2015), “P. obesi” (Mishra et al., 2013), “P. pacaensis” (Diop et al., 2016), “P. rachelemmaiella” (Hurst et al., 2022); “ P. grossensis” (Mishra et al., 2012), “P. phoceensis” (Mourembou et al., 2016), “P. raoultii” (Diop et al., 2016), “P. urinae” (Mbaye et al., 2022) and “P. vaginalis” (Diop et al., 2016), consist of effectively published but non-validated names. In earlier work on Peptoniphilus species, the members of this genus have been found to form multiple distinct clades in phylogenetic trees based on 16S rRNA gene sequences (Brown et al., 2014, Diop et al., 2019, Cho et al., 2015, Aujoulat et al., 2021;44(5):126235., Ryu et al., 2021, Rettenmaier et al., 2020), and the five originally described species generally grouped within different clades. Furthermore, in these studies, species from the genus Anaerosphaera and Aedoeadaptatus were also found to branch in between Peptoniphilus species (Johnson et al., 2014, Diop et al., 2019, Cho et al., 2015, Rettenmaier et al., 2020, Tomazetto et al., 2017), making this genus both polyphyletic and paraphyletic. An earlier study has also noted that the 16S rRNA sequence similarity between the type species P. asaccharolyticus and other examined Peptoniphilus species differed by 12.56 % (Aujoulat et al., 2021;44(5):126235.), which is much higher than the suggested differences in similarity values (≈ 5.0 %) for species within a genus (Yarza et al., 2014). This observation indicated that the Peptoniphilus species are genetically more diverse than a genus-level taxon (Aujoulat et al., 2021;44(5):126235.). Additionally, several non-validly published Peptoniphilus species viz. “P. pacaensis” (Diop et al., 2016), “P. phoceensis” (Mourembou et al., 2016), “P. raoultii” (Diop et al., 2016); “P. rachelemmaiella” (Hurst et al., 2022) and “P. vaginalis” (Diop et al., 2019), have been described only through genome sequence announcements and more information is needed to support their distinct species status. With the availability of whole genome sequences (WGS) (Sayers EW, Agarwala R, Bolton EE, Brister JR, Canese K et al. Database resources of the national center for biotechnology information. Nucleic Acids Res, 2019), delineation of different species as well as their phylogeny and taxonomy can now be more reliably determined based on different approaches using WGS-based analyses (Yarza et al., 2014, Hugenholtz et al., 2021, Gupta et al., 2020, Varghese et al., 2015, Kim et al., 2014, Richter and Rosselló-Móra, 2009, Goris et al., 2007). Parks et al. (Parks et al., 2018) have recently created a genome taxonomy database (GTDB) based on phylogenetic analysis of 120 ubiquitous single-copy proteins. The GTDB uses relative evolutionary divergence of different taxa in their protein tree for the delineation of taxa of different ranks, and it has now become an important resource for understanding prokaryotic taxonomy (Hugenholtz et al., 2021). In the GTDB taxonomy, species from the genus Peptoniphilus are placed into at least four novel genera in addition to the genus Peptoniphilus sensu stricto (Parks et al., 2018). All these observations highlight the need for developing a more reliable and informative classification scheme for the Peptoniphilus species.

Genome sequences are now available for 21 (of 23) validly named Peptoniphilus species and all 10 non-validly published species in the NCBI database (Sayers EW, Agarwala R, Bolton EE, Brister JR, Canese K et al. Database resources of the national center for biotechnology information. Nucleic Acids Res, 2019). In addition, genome sequences are also available for representative species from most other genera within the family Peptoniphilaceae. Using these genomes, which provide comprehensive coverage of the genetic diversity within the genus Peptoniphilus, in this study we have comprehensively examined the evolutionary relationships among Peptoniphilus species and related genera using multiple independent approaches. The approaches we have used include: (a) Construction of phylogenomic trees based on the concatenated sequences of core genome proteins, and another tree based on 120 ubiquitous single-copy proteins used in construction of the bac120 GTDB tree (Parks et al., 2018); (b) Construction of a tree based on 16S rRNA gene sequences for the type strains of all Peptoniphilaceae species; (c) Determination of relatedness between different species based on different sequence based matrices including pairwise average nucleotide identity (ANI) (Kim et al., 2014, Konstantinidis and Tiedje, 2005), digital DNA-DNA hybridization (dDDH) (Goris et al., 2007, Meier-Kolthoff et al., 2013), 16S rRNA sequence similarity (Kim et al., 2014, Stackebrandt et al., 2002); average amino acid identity (AAI) (Konstantinidis and Tiedje, 2007), and percentage of conserved proteins (POCP) (Qin et al., 2014); (d) Detailed analyses of proteins sequences from these species to identify molecular markers consisting of conserved signature indels (CSIs) (Gupta et al., 2020, Naushad et al., 2014, Gupta et al., 2014), which are uniquely found in species from different observed genus-level clades of Peptoniphilus species. Results from these different investigations provide strong and consistent evidence that the known Peptoniphilus species group into seven genus level clades (taxa) in addition to the genus Peptoniphilus sensu stricto. Species from all observed clades can be reliably distinguished from each other based on multiple exclusively shared molecular signatures that robustly demarcate these species clades in molecular terms. Based on the strong evidence presented here, we are proposing the transfer of several Peptoniphilus species which group reliably within the Aedoeadaptatus clade, that contains the type species of the genus Aedoeadaptatus, into the emended genus Aedoeadaptatus. The results presented here also demonstrate the usefulness of the described molecular markers for the placement of several uncharacterized Peptoniphilus spp. (both cultured and uncultured) into the indicated genus level clades. These results showing the existence and reliable demarcation of different genus level clades of Peptoniphilus species should prove very useful in more clearly understanding the roles of Peptoniphilus species from specific clades in different diseases.