Introduction
============

The majority of the *Campylobacter* species are associated with endothermic mammalian and avian hosts. Recently, a novel *Campylobacter* species has been described, *Campylobacter iguaniorum* (*Cig*), which is predominantly isolated from ectothermic reptilian hosts ([@evw218-B14]). Chelonians and lizards in particular appear to be a primary reservoir of this *Campylobacter* species. Reported overall prevalence in reptiles based on culturing was 8.2%; 15.6% of the chelonians and 6.1% of the lizards, but none of the snakes examined, carried this *Campylobacter* species ([@evw218-B12]). Recently, *Cig* has also been isolated from a non-reptilian host ([@evw218-B25]).

Within the genus *Campylobacter*, this species forms a clearly separated phylogenetic clade, together with the closely related taxa *C*. *fetus*, *C*. *hyointestinalis*, and *C*. *lanienae* (collectively called the *C*. *fetus* clade). *C*. *fetus* currently comprises three subspecies: mammal-associated *C*. *fetus* subsp. *fetus* (*Cff*) and *C*. *fetus* subsp. *venerealis* (*Cfv*), and reptile-associated *C*. *fetus* subsp. *testudinum* (*Cft*) ([@evw218-B10]). As with *Cig*, *Cft* has been shown to be associated primarily with reptiles, and both taxa are the most frequently isolated *Campylobacter* in reptiles ([@evw218-B19]; [@evw218-B38]; [@evw218-B12]). Also, *C*. *hyointestinalis* has been infrequently isolated from reptiles ([@evw218-B12]). Interestingly, *Campylobacter* species commonly found in various avian and mammalian hosts, such as *C*. *coli*, *C*. *jejuni*, and *C*. *lari*, were not isolated from reptiles, despite culturing conditions suitable for these species. It was speculated that the host body temperature, which is on average lower and more fluctuating in reptiles than in mammals and birds, is associated with this remarkable species distribution ([@evw218-B12]). Indeed, *Cig*, *C*. *fetus*, and to a lesser extent *C*. *hyointestinalis*, were found able to grow at lower temperatures than most other *Campylobacter* species ([@evw218-B14]).

The pathogenicity of *Cig* in reptiles is unknown; isolates have been recovered both from reptiles with and without clinical signs of disease ([@evw218-B2]; [@evw218-B12]). In contrast to reptile-associated *Cft*, which has been shown to cause infection in humans ([@evw218-B37]; [@evw218-B28]), no human *Cig* infections have been reported to date.

The first closed and annotated *Cig* genome has been described previously ([@evw218-B13]). Genome analysis and comparison provide valuable insights into host adaptation, virulence, phylogeny, and evolution of this reptile-associated *Campylobacter* species. Comparison with reptile-associated *Cft* could identify factors specific for adaptation to their shared reptilian host. Here we report the complete whole genome sequences of two *Cig* strains isolated from reptiles, and compare them to those of the closely related taxa *Campylobacter fetus*, *C*. *hyointestinalis*, and *C*. *lanienae*.

*Campylobacter iguaniorum* genome features and comparison
---------------------------------------------------------

The genome size of strain 1485E is 1,684,608 bp with a 70,030 bp megaplasmid; strain 2364D is 1,809,624 bp with an estimated 54,764 bp megaplasmid. The general features of *Cig* strains 1485E and 2463D are summarized in [supplementary table S1](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online.

Multiple genomic regions were specific for *Cig*, i.e. no orthologs were identified in all or most members of the *C*. *fetus* clade or the entire *Campylobacter* genus. In total, 59 genes were conserved in both *Cig* strains, which were absent from all other *C*. *fetus* clade members ([supplementary table S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). Multiple genes involved in sulfur metabolism were identified, indicating that this is important in *Cig* biology. Indeed, phenotypic testing showed that *Cig* and *C*. *hyointestinalis* are one of the few *Campylobacter* species that produce H~2~S on sulfate containing TSI agar ([@evw218-B14]). Notably, the gene coding for [l]{.smallcaps}-lactate permease (*lctP*) was absent from *Cig* and *C*. *hyointestinalis* subsp. *hyointestinalis*, but was well conserved in the other *C*. *fetus* clade members and most other *Campylobacter* species. In *C*. *jejuni*, transport of [l]{.smallcaps}-lactate occurs primarily via LctP, although at least one other transport route also exists ([@evw218-B36]). This predicts that *Cig* relies less on exogenous [l]{.smallcaps}-lactate as carbon or energy source than other *Campylobacter* species encoding *lctP*.

Several genes were found to be shared specifically by both reptile-associated taxa *Cig* and *Cft* ([supplementary table S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). As in reptile-associated *Cft*, a putative tricarballylate catabolism locus *tcuRABC* (CIG1485E_0479-0482; CIG2463D_0480-0483) was identified in both *Cig* strains. This locus is partially present in *C*. *hyointestinalis* subsp. *lawsonii*, but as *tcuC* is a truncated pseudogene, it is likely not functional. Tricarballylate could potentially be used as a carbon and energy source by *Cig*, as this locus has been shown to function in the catabolism of tricarballylate (a citrate analog) ([@evw218-B21]). Tricarballylate is considered to cause grass tetany in ruminants, a disease characterized by acute magnesium deficiency. In the ruminant rumen, *trans*-aconitate present in grass is rapidly reduced to tricarballylate, a toxic end product of ruminal fermentation ([@evw218-B32]). Neither the ruminant nor the normal rumen flora can catabolize tricarballylate efficiently. However, it has been shown that *Salmonella enterica* serovar Typhimurium strain LT2 can use tricarballylate as a carbon and energy source; the end product of tricarballylate metabolism is *cis*-aconitate, which then enters into the citric acid cycle ([@evw218-B18]; Lewis et al. [@evw218-B21]). Conservation of the *tcuRABC* locus in the reptile-associated taxa suggests that tricarballylate is ubiquitous in the niche inhabited by *Cig* and *Cft*, i.e., the mucosa of the reptilian intestines. As the *tcuRABC* locus is present in all highly prevalent reptile-associated *Campylobacter* taxa, this locus could confer an advantage in colonization of the reptilian host.

In contrast to most *Campylobacter* species, both *Cig* strains lack a defined lipooligosaccharide (LOS) region, bounded by *waa* genes and containing multiple glycosyltransferases ([supplementary table S2](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). The lack of *waaDEF*, which are highly conserved in *Campylobacter* and *Arcobacter*, is especially remarkable. Instead, multiple separate predicted glycosylation regions were identified ([fig. 1](#evw218-F1){ref-type="fig"}). A large glycosylation region (\>50 kb) was present in both strains. Although syntopic in both strains, large parts of this region were not homologous and, in combination with the deviant G + C content (29.0--30.0%), suggests acquisition via lateral transfer. However, this region was highly homologous in strain 2463D and *C*. *hyointestinalis* subsp. *hyointestinalis*. In strains 1485E and 2463D, respectively 20.6% (7/34) and 22.0% (9/41) of the GC tracts were identified within this glycosylation region.

![BRIG plots of *C*. *iguaniorum* and most closely related *Campylobacter* species. BLASTN-based genomic comparison of *C*. *iguaniorum* strains 1485E and 2463D, *C*. *fetus* subsp. *testudinum* (Cft) strains 03-427 and SP3, *C*. *fetus* subsp. *fetus* (Cff) strain 82-40, *C*. *fetus* subsp. *venerealis* (Cfv) strain 97/608, *C*. *hyointestinalis* subsp. *hyointestinalis* (Chh) strain LMG 9260, *C*. *hyointestinalis* subsp. *lawsonii* (Chl) strain CCUG 27631, and *C*. *lanienae* (Clan) strain NCTC 13004. Characteristic features of *C*. *iguaniorum* have been highlighted. Reference genome is *C*. *iguaniorum* strain 1485E (*A*); reference genome is *C*. *iguaniorum* strain 2463D (*B*).](evw218f1p){#evw218-F1}

As in other *Campylobacter* species many of the hypervariable GC tracts reside in surface structure-related genes showing phase variation. However, in strains 1485E and 2463D, respectively 23.5% (8/34) and 20.0% (8/41) of the GC tracts were located within autotransporter domain-containing genes, whose role in *Cig* biology remains to be determined. In total, seven autotransporter domain-containing genes were found conserved in both strains and showed no or low homology with other *Campylobacter* species. Noteworthy, 89% (8/9) of the autotransporter domain-containing genes in both strains contained a GC tract.

The *cas* genes present in *Cig* were homologous with those in *C*. *hyointestinalis* subsp. *hyointestinalis*, but not with those in *C*. *fetus*, indicating that at least two different CRISPR/Cas systems are present within the *Campylobacter fetus* clade ([fig. 1](#evw218-F1){ref-type="fig"}).

One of the characteristics shared by *Cig*, *C*. *fetus*, and most *Arcobacter* species is the ability to grow at lower temperatures (≤25 °C) than most other *Campylobacter* species ([@evw218-B27]; [@evw218-B14]). Also, these species have been shown to occur together in reptilian hosts, which often show a broad body temperature range and on average lower body temperatures compared with most endothermic mammals and birds ([@evw218-B12]). As in *C*. *fetus*, but in contrast to *C*. *jejuni* in which *nuoE* and *nuoF* are lacking ([@evw218-B20]), all NADH:quinone oxidoreductase complex I subunits (NuoA-N) are present in *Cig*, suggesting that NADH is an important electron donor in this species. Interestingly, NuoA-N present in *Cig* and all other *C*. *fetus* clade members showed higher homology to species from the *Arcobacter* genus (≤72%), than to other species from the *Campylobacter* genus (≤55%). The high homology observed in the NADH:quinone oxidoreductase complex I subunits might be related to low temperature adaptation. Indeed, in other organisms NADH:quinone oxidoreductase complex I is considered the most thermolabile protein complex of oxidative phosphorylation ([@evw218-B5]). Within the *Campylobacter* genus, the ability to exclusively proliferate at lower temperatures (18--37 °C) is unique for *Cig*. This feature may have helped *Cig* colonize hosts which have a low and variable body temperature.

*Campylobacter iguaniorum* phylogeny and diversity
--------------------------------------------------

A phylogenomic reconstruction accounting for the effects of homologous recombination was performed, based on a 1,042,737 nucleotide gapless alignment of the whole genomes of *Cig* and most closely related species *C*. *fetus*, *C*. *hyointestinalis*, and *C*. *lanienae* ([supplementary fig. S1](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). *Cig* was clearly distinct from the other *Campylobacter* species. The first split occurred between *C*. *lanienae* and the other species. Excluding the more distantly related *C*. *lanienae*, the branch lengths indicated that *Cig* is less related to the last common ancestor than the other species. Interestingly, *Cig*, *C*. *fetus*, and *C*. *hyointestinalis* branch of the last common ancestor at the same point, suggesting that these species started diverging at the same time.

In order to examine the genomic relatedness in further detail, the average nucleotide identity (ANI) was determined for the whole genomes of both *Cig* strains and strains of the most closely related taxa *C*. *fetus*, *C*. *hyointestinalis*, and *C*. *lanienae* ([supplementary table S3](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). ANI values were highest for *Cig* and both *C*. *hyointestinalis* and *C*. *fetus*. Homology was higher between *Cig* and reptile-associated *Cft* than between *Cig* and mammal-associated *C*. *fetus* subspecies *fetus* and *venerealis*, indicating that both reptile-associated taxa *Cig* and *Cft* share genomic regions, which might be associated with adaptation to their shared reptilian hosts. Higher ANI values between *Cig* strain 2463D and *Cft* strain SP3, *C*. *fetus* subsp. *venerealis*, and *C*. *hyointestinalis* subsp. *hyointestinalis* can be explained by shared laterally transferred genomic regions, such as prophages, and shared glycosylation regions ([fig. 1](#evw218-F1){ref-type="fig"}). In addition to this, the genes shared by *Cig* and other members of the *C*. *fetus* clade (≥50% identity) showed that most genes were shared between *Cig* and *C*. *hyointestinalis* subsp. *hyointestinalis*, followed by both *Cft* strains, suggesting that these taxa are most closely related ([supplementary table S4](#sup1){ref-type="supplementary-material"}, [Supplementary Material](#sup1){ref-type="supplementary-material"} online). Nevertheless, on a species level, differences based on shared genes and ANI are small and in support of the whole genome-based phylogeny showing similar divergence between *Cig*, *C*. *fetus*, and *C*. *hyointestinalis*.

MLST analysis of 18 *Cig* strains showed two distinct lineages and a high intraspecies diversity without any identical sequence type ([fig. 2](#evw218-F2){ref-type="fig"}). No clear association with host type was observed, as *Cig* isolates originating from lizards and chelonians were found in both lineages. Nevertheless, host association was observed at the species level to some degree, as *Cig* isolates originating from animals of the same species, but from different locations were mostly clustering together. This was most clear in *Iguana iguana*, in which two *Cig* isolates, which were obtained from different animals in 2003 and 2012, clustered closely together. In contrast, two isolates obtained from the same animal (11S02571-1 and 11S02571-4) showed a large genetic distance, indicating that *Cig* diversity within the same animal can be high.

![Maximum likelihood dendrogram of *C*. *iguaniorum* based on concatenated MLST sequences, using 500 bootstraps. Isolate numbers are followed by the host species.](evw218f2p){#evw218-F2}

Both *Cig* and *Cft* colonize the same reptilian hosts ([@evw218-B12]), facilitating potential lateral gene transfer between these two taxa. Indeed, both reptile-associated taxa share specific genomic regions which might confer certain competitive advantages to survive in the reptilian host. Furthermore, similar prophages were identified in the accessory genomes of both taxa, which can serve as a vehicle for novel genetic material and enable gene flow between both taxa. However, no recent recombination events between both taxa were detected in the core genomes. Despite the close genetic relationship and shared host type, the recombination rate between both reptile-associated taxa can be considered low compared with certain other *Campylobacter* species colonizing a shared host ([@evw218-B35]). This could be explained by a recent introduction in the same host or by barriers such as an intrinsic resistance to interspecific recombination or separated niches within the host, as has been shown for *C*. *jejuni* ([@evw218-B34]). Instead, multiple regions showed higher than expected homology in *C*. *iguaniorum*, *C*. *hyointestinalis*, and members of the *C*. *concisus* clade, suggesting gene flow between these distantly related *Campylobacter* taxa.

Material and Methods
====================

Strains
-------

Characteristics of all strains used in this study are summarized in [table 1](#evw218-T1){ref-type="table"}. *Cig* strain 1485E (= CCUG 66346 = LMG 28143) was isolated in 2003 from a bearded dragon (*Pogona vitticeps*) with a hypertrophic and perforated colon. *Cig* strain 2463D (= CCUG 66347) was isolated in 2003 from a green iguana (*Iguana iguana*) with chronic interstitial nephritis. All strains were grown on Columbia agar with 5% sheep blood (Oxoid, the Netherlands) in a microaerobic atmosphere (83.3 N~2~, 7.1% CO~2~, 3.6% H~2~, and 6% O~2~) at 37 °C for 48 h.

###### 

Features of the *Campylobacter* Strains Used in this Study

  Species   Strain                   Source organism             Source type      Location   Sequence data       Sequence method     Reference                                  Accession number
  --------- ------------- -------------------------------------- ---------------- ---------- --------------- ----------------------- --------------- ----------------------------------------------------------------------
  Cig       1485E              Lizard (*Pogona vitticeps*)       Feces            NL         WGS              454, Illumina, PacBio  [@evw218-B13]                             CP009043-CP009044
  Cig       2463D                Lizard (*Iguana iguana*)        Feces            NL         WGS              454, Illumina, PacBio  [@evw218-B14]                                  CP010995
  Cig       11S02571-1       Chelonian (*Chelodina mccordi*)     Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697811, KU697824, KU697837, KU697850, KU697863, KU697876, KU697889
  Cig       11S02571-4       Chelonian (*Chelodina mccordi*)     Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697812, KU697825, KU697838, KU697851, KU697864, KU697877, KU697890
  Cig       11S02590-1       Lizard (*Pogona henrilawsonii*)     Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697813, KU697826, KU697839, KU697852, KU697865, KU697878, KU697891
  Cig       11S03163-2     Chelonian (*Agrionemys horsfieldii*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697814, KU697827, KU697840, KU697853, KU697866, KU697879, KU697892
  Cig       12S01208-3     Chelonian (*Chelonoidis carbonaria*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697815, KU697828, KU697841, KU697854, KU697867, KU697880, KU697893
  Cig       12S02279-8     Chelonian (*Aldabrachelys gigantea*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697816, KU697829, KU697842, KU697855, KU697868, KU697881, KU697894
  Cig       12S02285-1          Lizard (*Sauromalus ater*)       Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697817, KU697830, KU697843, KU697856, KU697869, KU697882, KU697895
  Cig       12S02360-1           Lizard (*Iguana iguana*)        Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697818, KU697831, KU697844, KU697857, KU697870, KU697883, KU697896
  Cig       12S02842-24    Chelonian (*Aldabrachelys gigantea*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697819, KU697832, KU697845, KU697858, KU697871, KU697884, KU697897
  Cig       12S03949-1     Lizard (*Hemitheconyx caudicinctus*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697820, KU697833, KU697846, KU697859, KU697872, KU697885, KU697898
  Cig       12S05338-1      Chelonian (Stigmochelys pardalis)    Feces            FR         MLST                    Sanger          [@evw218-B2]     KU697821, KU697834, KU697847, KU697860, KU697873, KU697886, KU697899
  Cig       13S00387-3     Chelonian (*Chelonoidis carbonaria*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697822, KU697835, KU697848, KU697861, KU697874, KU697887, KU697900
  Cig       13S00406-3     Chelonian (*Agrionemys horsfieldii*)  Feces            NL         MLST                    Sanger          [@evw218-B12]    KU697823, KU697836, KU697849, KU697862, KU697875, KU697888, KU697901
  Cff       82-40                         Human                  Blood            US         WGS                     Sanger          [@evw218-B29]                                  CP000487
  Cft       03-427                        Human                  Blood            US         WGS              454, Illumina, PacBio  [@evw218-B11]                                  CP006833
  Cft       SP3                Snake (*Heterodon nasicus*)       Feces            UK         WGS              454, Illumina, PacBio  [@evw218-B15]                                  CP010953
  Cfv       97/608                        Bovine                 Placenta         AR         WGS              454, Illumina, PacBio  [@evw218-B17]                             CP008810-CP008812
  Chh       LMG 9260                      Human                  Feces            BE         WGS                  454, Illumina      [@evw218-B26]                                  CP015575
  Chl       CCUG 27631                   Porcine                 Gastric biopsy   SE         WGS                  454, Illumina      [@evw218-B26]                                  CP015576
  Clan      NCTC 13004                    Human                  Feces            SE         WGS                       454           [@evw218-B22]                                  CP015578

Cig, C. iguaniorum; Cff, C. fetus subsp. fetus; Cft, C. fetus subsp. testudinum; Cfv, C. fetus subsp. venerealis; Chh, C. hyointestinalis subsp. hyointestinalis; Chl, C. hyointestinalis subsp. lawsonii; Clan, C. lanienae.

AR, Argentina; BE, Belgium; FR, France; NL, Netherlands; SE, Sweden; UK, United Kingdom; US, United States. MLST, multilocus sequence typing; WGS, whole genome sequencing.

Whole Genome Sequencing
-----------------------

The sequencing of *Cig* strains 1485E and 2463D was performed using shotgun and paired-end reads obtained on a Roche 454 FLX Genome Sequencer. A total of 292,658 (1485E) or 318,374 (2463D) 454 reads were assembled using the Newbler assembler (v2.6) into single chromosomal scaffolds of 12 (1485E) or 11 (2463D) unique contigs and single megaplasmid scaffolds, providing draft genome sequences with coverages of 68× (1485E) or 65× (2463D). All 454 base calls were validated using 1,570,644 (1485E) or 2,300,370 (2463D) Illumina MiSeq reads, providing an additional 178× (1485E) or 263× (2463D) coverage. Scaffold gaps were filled as described ([@evw218-B23]). Sequences across the contig junctions were confirmed with Sanger sequencing. Assembly was confirmed using PacBio long reads for strain *Cig* 2463D. PacBio RS reads were assembled into contigs using Quiver (Pacific Bioscience, Menlo Park, CA, USA). Homopolymeric GC tracts were characterized using the high-depth MiSeq reads.

Genome Analysis
---------------

Protein-, rRNA-, and tRNA-encoding genes were identified as described ([@evw218-B23]). The 1485E genome was annotated based on *Cft* strain 03-427^T^ (accession number CP006833) ([@evw218-B11]; [@evw218-B10]) and the annotation of the 2463D genome was based on that of 1485E, with further annotation using Artemis ([@evw218-B33]), the identification of Pfam domains (v.27.0) ([@evw218-B9]), and BLASTP comparisons to proteins in the NCBI non-redundant (nr) database. CRISPR regions were identified using CRISPRFinder ([@evw218-B16]). The complete annotated genome sequence of *Cig* strain 1485E has been deposited in GenBank under accession numbers CP009043 (chromosome) and CP009044 (megaplasmid) ([@evw218-B13]). The complete annotated genome sequence of the *Cig* strain 2463D chromosome has been deposited in GenBank under accession number CP010995. Accession numbers of all genomes used in this study can be found in [table 1](#evw218-T1){ref-type="table"}.

The *C*. *iguaniorum* strain 1485E genome was compared with the genome of strain 2463D and the completed genomes of the closely related taxa *C*. *fetus* subsp. *testudinum* (strains 03-427 and SP3), *C*. *fetus* subsp. *fetus* (strain 82-40), *C*. *fetus* subsp. *venerealis* (strain 97/608), *C*. *hyointestinalis* subsp. *hyointestinalis* (strain LMG 9260), *C*. *hyointestinalis* subsp. *lawsonii* (strain CCUG 27631), and *C*. *lanienae* (NCTC 13004). For *Campylobacter* species outside the *C*. *fetus* clade, BLASTP comparisons to proteins in the NCBI non-redundant (nr) database were performed. A local BLAST was performed based on the predicted proteomes of all genomes and the results were screened for features specific for one or both *Cig* strains and the other members of the *C. fetus* clade. Using JSpecies v.1.2.1 ([@evw218-B31]) and BLAST v.2.2.26, average nucleotide identity (ANI) values based on the whole genome sequences were calculated for these strains as a measure of genetic relatedness. The BLAST parameters were: X = 150, q = −1, F = F, e = 1e−15, and a = 2. To visualize genomic regions specific for *Cig*, the BLAST ring image generator (BRIG) ([@evw218-B1]) was used, based on BLAST v.2.2.26.

Orthologous Grouping and Phylogenomic Reconstruction
----------------------------------------------------

An all versus all BLAST was performed for all predicted proteins of the whole genomes ([table 1](#evw218-T1){ref-type="table"}) at an E-value cutoff of 1E−6. To determine the orthologous relationships of all proteins, the BLAST output was parsed by Orthagogue ([@evw218-B7]). Proteins were considered for orthology clustering if the proteins had at least 50% identity and at least 50% overlap. To determine the orthologous groups, Markov clustering (MCL) was performed using MCL-edge ([@evw218-B8]). Genes encoding the proteins were aligned with each other within their respective orthologous groups using MUSCLE ([@evw218-B6]). A super alignment was created by concatenating the aligned genes according to their position in *Cig* strain 1485E if they were present in all isolates. Gaps were removed using Gblocks ([@evw218-B3]). Based on this super alignment phylogenomic reconstruction and prediction of recombination events was performed using Gubbins ([@evw218-B4]) with the default settings. Phylogenetic dendrograms were created using Fasttree ([@evw218-B30]). A BLAST search of the predicted recombination regions of both *Cig* strains against the genes of *Cft* was performed to search for particular recombination between these reptile-associated taxa.

MLST
----

Multilocus sequence typing (MLST) was performed for all *Cig* strains listed in [table 1](#evw218-T1){ref-type="table"}. The MLST loci were extracted from the genomes of 1485E and 2463D and sequenced as described previously for the other *Cig* isolates ([@evw218-B24]). For HFglt, the annealing temperature was lowered to 46 °C and 40 instead of 35 cycles were used. Sequences were trimmed and concatenated. In MEGA v.6.06, the concatenated sequences were aligned using MUSCLE and a Maximum likelihood dendrogram was created using 500 bootstraps. Accession numbers of the MLST sequence data can be found in [table 1](#evw218-T1){ref-type="table"}.

Supplementary Material
======================

###### 

Click here for additional data file.

We thank Linda van der Graaf-van Bloois for valuable advice and Arjen Timmerman for technical support. We thank Mary Chapman and Nathaniel Simon for the generation of Illumina MiSeq reads and assistance in the final assembly and genome closure. This work was partially supported by USDA-ARS CRIS project 5325-42000-047-00D.

[^1]: **Associate editor**: Howard Ochman
